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WO2025155821A1 - Procédés, kits, systèmes et compositions pour la détection multiplexe de cibles - Google Patents

Procédés, kits, systèmes et compositions pour la détection multiplexe de cibles

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Publication number
WO2025155821A1
WO2025155821A1 PCT/US2025/012034 US2025012034W WO2025155821A1 WO 2025155821 A1 WO2025155821 A1 WO 2025155821A1 US 2025012034 W US2025012034 W US 2025012034W WO 2025155821 A1 WO2025155821 A1 WO 2025155821A1
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WIPO (PCT)
Prior art keywords
nucleic acid
target
labeled
binding ligand
acid probe
Prior art date
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English (en)
Inventor
Kuanwei SHENG
Peng Yin
Hanquan SU
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Harvard University
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Harvard University
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Publication of WO2025155821A1 publication Critical patent/WO2025155821A1/fr
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    • 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
    • C12Q1/6841—In situ hybridisation

Definitions

  • the first or second target-binding ligand comprises a protein.
  • the first or second target-binding ligand comprises RNA.
  • the first or second nucleic acid tag is DNA
  • nuclease is a DNase
  • the first nucleic acid tag is DNA conjugated with the antibody or antigen-binding fragment thereof.
  • the first nucleic acid tag is DNA conjugated with an RNA.
  • the nuclease is an RNase.
  • the second nucleic acid tag is DNA conjugated with protein.
  • the second nucleic acid tag is DNA conjugated with an antibody or antigen-binding fragment thereof.
  • the step of contacting the preparation of cells or tissue with the second target-binding ligand comprises: (i) contacting the preparation of cells or tissue with a second target-binding ligand conjugated to a second oligonucleotide primer, under conditions permitting specific binding of the second target-binding ligand to the target molecule; (ii) adding to the preparation of cells or tissue a reaction mixture comprising a second singlestranded extender template and a nucleic acid polymerase enzyme under conditions permitting hybridization and extension of the second oligonucleotide primer using the second singlestranded extender template; (iii) heating the reaction mixture of step (ii) to separate the second single-stranded extender template from extended second oligonucleotide primer produced in step (ii); and (iv) repeating steps (ii) and (iii) at least once, thereby generating a concatemer comprising repeats of the second oligonucleotide primer sequence conjug
  • the first nucleic acid tag is a branched nucleic acid, optionally, the first nucleic acid tag is a branched DNA.
  • a plurality of labeled nucleic acid probe molecules hybridizes to first or second nucleic acid tag.
  • a single labeled nucleic acid probe molecules hybridizes to first or second nucleic acid tag.
  • a concentration of the blocking probes is higher than a concentration of the labeled-nucleic acid probes. In some other embodiments, a concentration of the blocking probes is lower than a concentration of the labeled-nucleic acid probes.
  • each nucleic acid tag is a concatemer comprising repeats of a respective first oligonucleotide primer sequence.
  • the step of contacting the preparation of cells or tissue with a set of target-binding ligand molecules comprises: (i) contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective first oligonucleotide primer members of a set of orthogonal first oligonucleotide primer and first single-stranded extender template oligonucleotide pairs, wherein the first oligonucleotide primer of each pair has a different sequence from other first oligonucleotide primers in the set, and is conjugated to a different member of the set of target-binding ligand molecules; (ii) contacting the preparation of cells or tissue with the set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to
  • each nucleic acid tag is a branched nucleic acid, optionally, the branched nucleic acid is DNA.
  • each target-binding ligand comprises a protein.
  • each target-binding ligand comprises an antibody or antigen-binding fragment thereof.
  • each target-binding ligand comprises RNA.
  • a plurality of labeled nucleic acid probe molecules hybridizes to a plurality of concatemer repeats conjugated to the target-binding ligands.
  • the preparation of cells or tissue is fixed.
  • the preparation of tissue is paraffin embedded.
  • one multiplex approach uses a set of orthogonal target ligand-binding ligands, each specific for a different target of interest, and each conjugated to a different nucleic acid tag.
  • the respective nucleic acid tags provide different, highly selective binding site(s) for a set of different, distinguishably-labeled probes that permit multiplex detection, quantitation and/or imaging of the members of a set of target molecules in a sample at each detection step.
  • kits comprising at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, targetbinding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.
  • the kit comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe; and (v) at least one of: (a) a nuclease; and (b) a set of blocking probes.
  • a system for detecting targets in a sample comprising at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.
  • the system comprises: (i) a targetbinding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe; and (iii) at least one of: (a) a nuclease; and (b) a set of blocking probes.
  • the system comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe; and (v) at least one of: (a) a nuclease; and (b) a set of blocking probes.
  • Members of the second set of blocking-nucleic acid probes have a different nucleotide sequence from other members of the second set of blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules.
  • the complementary sequence of each blocking probe in the second set of blocking probes is of sufficient length to allow the blocking probe to bind, interact or hybridize with the nucleic acid tag conjugated to a target-binding ligand and inhibit or reduce binding, interaction or hybridization of the second labeled-nucleic acid probe to said nucleic acid tag.
  • the step of removing the blocking probes and labeled-nucleic acid from the nucleic acid tags and/or the sample comprises contacting the sample with an agent that dissociates hybridized nucleic acid strands, nucleic acid strands.
  • the sample can be contacted with a formamide solution (e.g., 50% formamide in IxPBS) which can dissociate the blocking probes and the labeled-nucleic acid probes from the nucleic acid tags.
  • Embodiments of the various aspects described herein include target binding ligands.
  • target-binding ligand target-binding ligand molecule
  • target-binding molecule target-binding molecule
  • a target-binding ligand or molecule is a molecule that is binds, e.g., e.g., specifically binds, to a target molecule of interest.
  • the targeting binding ligand can be a natural or synthetic molecule.
  • a target-binding ligand can be a biomolecule, such as a polypeptide or a polynucleotide.
  • a targetbinding ligand is a polypeptide.
  • exemplary target-binding ligands include, but are not limited to, peptides, polypeptides, antibodies, antigen binding fragment of antibodies, antibody derivatives, antigens, receptors, a ligand for a receptor, oligonucleotides, and polynucleotides.
  • a target-binding ligand can be selected from antibodies, antigen binding fragments of antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, nucleic acids (e.g., RNA and aptamers such as RNA or DNA aptamers), protein, peptide, binding partner, oligosaccharides, polysaccharides, lipopolysaccharides, cellular metabolites, cells, viruses, subcellular particles, haptens, pharmacologically active substances, alkaloids, steroids, vitamins, amino acids, avimers, peptidomimetics, hormone receptors, cytokine receptors, synthetic receptors, sugars or molecularly imprinted polymer.
  • nucleic acids e.g., RNA and aptamers such as RNA or DNA aptamers
  • protein e.g., RNA and aptamers such as RNA or DNA aptamers
  • protein e.g., RNA and aptamers
  • the target-binding ligand can be selective to a specific target or class of targets such as toxins and biomolecules.
  • targets such as toxins and biomolecules.
  • the target can be ions, molecules, oligomers, polymers, proteins, peptides, nucleic acids, toxins, biological threat agents such as spore, viral, cellular and protein toxins, carbohydrates (e.g., mono-saccharides, disaccharides, oligosaccharides, polyols, and polysaccharides) and combinations of these (e.g., copolymers including these).
  • the target-binding ligand is an antibody or antigen binding fragment thereof.
  • antibody refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence that binds a target molecule.
  • an antibody can include an immunoglobulin heavy (H) chain variable region (abbreviated herein as VH), and an immunoglobulin light (L) chain variable region (abbreviated herein as VL).
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • An antibody can include the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).
  • the term antibody as used herein includes any of a number of different constructs using one or more antigen-binding domains or fragments of an antibody to mediate binding to a target molecule.
  • an antibody in addition to a complete IgA, IgG, IgE, IgD or IgM antibody, an antibody includes, but is not limited to antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, domain antibodies (dAb) (de Wildt et al., Eur J Immunol. 1996; 26(3):629- 39) and nanobodies.
  • An affibody which uses a non-antibody scaffold to support a diverse target-binding domain, can also be used as a target-binding ligand in the methods, compositions and kits described herein.
  • antibody and “antibodies” include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab)2 fragments.
  • antibody and “antibodies” include intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies.
  • an “antigen-binding fragment” refers that portion of an antibody that is necessary and sufficient for binding to a given antigen.
  • an antigen binding fragment of a conventional antibody will comprise six complementarity determining regions (CDRs) derived from the heavy and light chain polypeptides of an antibody arranged on a scaffold that permits them to selectively bind the antigen.
  • CDRs complementarity determining regions
  • a commonly used antigen-binding fragment includes the VH and VL domains of an antibody, which can be joined either via part of the constant domains of the heavy and light chains of an antibody, or, alternatively, by a linker, such as a peptide linker.
  • Non-conventional antibodies such as camelid and short antibodies have only heavy chain sequences, denoted, for example VHH. These can be used in a manner analogous to VH/VL-containing antigen-binding fragments.
  • Non-limiting examples of antibody fragments encompassed by the term antigen-binding fragment include: (i) a Fab fragment, having VL, CL, VH and CHI domains; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) an Fd fragment having VH and CHI domains; (iv) a Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) an Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)) which consists
  • linear antibodies comprising a pair of tandem Fd segments (VH-CH1-VH- CHI) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870).
  • the molecules of Fv, scFv or diabody can be stabilized by incorporating disulfide bridges linking the VH and VL domains.
  • Minibodies comprising a scFv fragment linked to a CH3 domain can also be obtained.
  • binding fragments are Fab’, which differs from Fab fragments by the addition of some residues at the carboxyl terminus of the CHI domain of the heavy chain, including one or more cysteines of the hinge region of the antibody, and Fab’-SH, which is a Fab’ fragment in which the cysteine residue(s) of the constant domains carries a free thiol group.
  • the binding fragments can be produced by recombinant DNA techniques.
  • the binding fragments can also be produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies.
  • the target binding ligand can be a nucleic acid, e.g., the target binding ligand can be RNA.
  • the target binding ligand can be an RNA.
  • the binding of the target-binding ligand to the target molecule is a specific binding.
  • specific binding refers to a physical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a nontarget.
  • a reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
  • the specificity of a target-binding ligand can be determined based on affinity and/or avidity.
  • the affinity represented by the equilibrium constant for the dissociation (KD) of a target-binding ligand and its target, is a measure of the binding strength between the targetbinding ligand and its target. The lower the value of the KD, the stronger the binding strength between the target-binding ligand and its target.
  • the affinity can also be expressed as the affinity constant (KA), which is 1/ KD).
  • a target-binding ligand as described herein is said to be "specific for” or to “specifically bind” or “selectively bind” a first target compared to a second target when it binds to the first antigen with an affinity (as described above, and suitably expressed, for example as a KD value) that is at least 1000 times, 10000 times or more better than the affinity with target-binding ligand binds to another given molecule.
  • a molecule that “specifically binds,” “selectively binds” or “is specific for” a given target will bind with a KD of 10-5 M (10000 nM) or less, e.g., 10-6 M, 10 7 M, 10 8 M, 10 9 M, 10-10 M, 10-11 M, 10-12 M, or less.
  • Specific binding can be influenced by, for example, the affinity and avidity of the target-binding ligand and the concentration of the target-binding ligand.
  • the person of ordinary skill in the art can determine appropriate conditions under which a target-binding ligand selectively binds the target using any suitable methods, such as titration of in a suitable binding assay.
  • Antibody dissociation constants and affinities can be determined, for example, by a surface plasmon resonance-based assay (such as the BIAcore assay; Forte Bio OctetTM analysis, enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA’s), for example.
  • a surface plasmon resonance-based assay such as the BIAcore assay; Forte Bio OctetTM analysis, enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA’s
  • the dissociation constant between the target-binding ligand and target molecule is at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or greater.
  • the specific binding refers to binding where the target-binding ligand binds to its target molecule without substantially binding to any other species in the sample/test solution.
  • the target binding ligand(s) used for detecting a first target molecule(s) can be different from the target binding ligand(s) used for detecting a second target molecule(s).
  • the target binding ligand(s) used for detecting a first target molecule(s) can be an RNA and the target binding ligand(s) used for detecting a second target molecule(s) can be a non-nucleic acid molecule, e.g., an antibody, an antigen binding fragment of an antibody, an antigen, a receptor, a ligand for a receptor, an enzyme, or other polypeptide or protein.
  • the target binding ligand(s) used for detecting a first target molecule(s) can be same as the target binding ligand(s) used for detecting a second target molecule(s).
  • the target binding ligand(s) used for detecting a first target molecule(s) and the target binding ligand(s) used for detecting a second target molecule(s) can be independently a non-nucleic acid molecule.
  • the target binding ligand(s) used for detecting a first target molecule(s) and the target binding ligand(s) used for detecting a second target molecule(s) can be independently an antibody, an antigen binding fragment of an antibody, an antigen, a receptor, a ligand for a receptor, an enzyme, or other polypeptide or protein.
  • the target-binding ligand(s) is added to the sample under conditions permitting specific binding of the target-binding ligand to its target molecule.
  • Target-binding ligands as described herein can be contacted a sample according to methods known in the art.
  • the target-binding ligand is an antibody or antigen-binding fragment thereof
  • methods widely applied in immunohistochemistry can be used to stain the sample for detection of the given target ligand.
  • the methods for contacting the sample with the target-binding ligand can parallel those used with, e.g., fluorescently labeled antibodies or antibody fragments.
  • the contacting or staining can comprise the addition of a set of target-binding ligands, each comprising a different nucleic acid tag.
  • the target-binding ligand is not an antibody or antigen-binding fragment thereof
  • methods for contacting a sample with the target-binding ligand can be adapted from those known in the art for the given ligand.
  • the target molecule is a DNA or RNA molecule comprising a given sequence
  • conditions as used, e.g., for in situ hybridization can be used to permit binding of the target-binding ligand oligonucleotide(s) to the target sequence(s).
  • a set of orthogonal target ligand-binding ligands each specific for a different target of interest, and each conjugated to a different nucleic acid tag, can be used.
  • the respective nucleic acid tags provide different, highly selective binding site(s) for a set of different, distinguishably-labeled probes that permit multiplex detection, quantitation and/or imaging of the members of a set of target molecules in a sample at each detection step.
  • nucleic acid tag conjugated with a nucleic acid tag.
  • nucleic acid barcode and DNA barcode are used interchangeably herein and refer to a nucleic acid sequence that can be used to unambiguously identify the target-binding ligand it is attached to.
  • a nucleic acid tag can be linear or branched. Accordingly, in some embodiments, a nucleic acid tag described herein is a linear nucleic acid. In some other embodiments, a nucleic acid tag described herein is a branched nucleic acid.
  • a nucleic acid tag described herein comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more sites for binding with a labeled-nucleic acid probe.
  • the nucleic acid tag is a concatemer.
  • the term “concatemer” refers to a nucleic acid molecule comprising two or more repeats of a given sequence in a head-to-tail, 5’ to 3’ orientation.
  • repeat units in a concatemer can be independently, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
  • the labeled-nucleic acid probe comprises a sequence complementary to at least a part of the sequence repeated in the concatemer.
  • nucleic acid tag when the nucleic acid tag is concatemer, the overall length of the t nucleic acid tag is dependent on the number of repeats and their length.
  • a nucleic acid tag when it is not a concatemer, it can be, for example, between 9-100 nucleotides in length, between 9-95 nucleotides in length, between 9-90 nucleotides in length, between 9-85 nucleotides in length, between 9- 80 nucleotides in length, between 9-75 nucleotides in length, between 9-70 nucleotides in length, between 9-65 nucleotides in length, between 9-60 nucleotides in length, between 9-55 nucleotides in length, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-25 nucleotides in length
  • 11-90 nucleotides in length between 11-80 nucleotides in length, between 11-70 nucleotides in length, between 11-60 nucleotides in length, between 11-50 nucleotides in length, between
  • 13-80 nucleotides in length between 13-70 nucleotides in length, between 13-60 nucleotides in length, between 13-50 nucleotides in length, between 13-40 nucleotides in length, between
  • 14-70 nucleotides in length between 14-60 nucleotides in length, between 14-50 nucleotides in length, between 14-40 nucleotides in length, between 14-30 nucleotides in length, between
  • nucleotides in length between 15-100 nucleotides in length, between 15-90 nucleotides in length, between 15-80 nucleotides in length, between 15-70 nucleotides in length, between 15-60 nucleotides in length, between 15-50 nucleotides in length, between 15-40 nucleotides in length, between 15-30 nucleotides in length, or between 15-20 nucleotides in length.
  • the oligonucleotide primer can be, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
  • a single-stranded extender template oligonucleotide as described herein has a 3’ modification or moiety (also referred to as a “stopper” moiety) that blocks or precludes template-dependent extension.
  • Such a modification can also be referred to as a chain terminator, and includes, for example, a dideoxy nucleoside, 3’-CPR II CPG, 3 ’-phosphate CPG, 5’-OMe-dT-CE Phosphoramidite, 5’-amino-dT-CE Phosphoramidite, 2’3’-ddA-CE Phosphoramidite, 2’3’-ddC-CE Phosphoramidite, 2’3-ddG-CE Phosphoramidite, 2’3’-ddT-CE Phosphoramidite, 3’-dA-CPG, 3’-ddC-CPG, 3’-dC-CPG, 3’-dG-CPG, 3’-dT-CPG, 3’-Spacer- C3 CPG and the like.
  • a single-stranded extender template oligonucleotide can be, for example, between 9- 100 nucleotides in length, between 9-95 nucleotides in length, between 9-90 nucleotides in length, between 9-85 nucleotides in length, between 9- 80 nucleotides in length, between 9-75 nucleotides in length, between 9-70 nucleotides in length, between 9-65 nucleotides in length, between 9-60 nucleotides in length, between 9-55 nucleotides in length, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-25 nucleotides in length, between 9-20 nucleotides in length, between 9-15 nucleotides in length, between 10-100 nucleotides in length, between 10-90 nucleot
  • the single-stranded extender template oligonucleotide can be, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
  • the single-stranded extender template and the nucleic acid polymerase enzyme are added to the sample under conditions permitting hybridization and extension of the oligonucleotide primer using the single-stranded extender template.
  • condition permitting hybridization and extension of a given molecule, e.g., an oligonucleotide molecule, refers to conditions of temperature, salt, buffer and other reaction components sufficient for a template-directed polymerase-mediated primer extension reaction. Exact conditions for extension by a given polymerase vary with the enzyme chosen, but are known in the art and/or can be determined without undue experimentation.
  • Conditions for hybridization e.g., of a primer as described herein to a single-stranded extender template as described herein, will generally be those salt, buffer and other reaction component conditions appropriate for the chosen polymerase enzyme, with the annealing or hybridization temperature established by one of skill in the art primarily on the basis of the length of the concatemer repeat unit and degree of multiplexing in a given reaction.
  • an annealing or hybridization temperature can be determined based on the Tm of the specific concatemer repeat sequence - generally the annealing temperature in a cycling reaction is about 5°C below the Tm for the hybridization of the repeat sequence to its complement under the salt and reaction component concentrations optimal for the enzyme of choice.
  • the length of the repeat unit takes on added importance, as the Tm for various repeat sequences will vary.
  • Primer/single-stranded extended template design can take sequence variation into account to design primers and single-stranded extender templates that have Tm values that are relatively close to each other, generally on the order of within 5-7°C for all members of a set of primer/extender template sequences. Under these circumstances, a single annealing or hybridization temperature in a cycling reaction can permit efficient hybridization and extension of members of the set in the multiplex.
  • the reaction can be incubated at an annealing temperature for a period of time (generally a matter of seconds to minutes) before raising the temperature to an optimum extension temperature for the polymerase enzyme. It should be understood that contacting a nucleic acid molecule with a single-stranded extender template “under conditions permitting hybridization and extension” of the nucleic acid molecule can include such an annealing/extending temperature shift.
  • annealing or hybridization occurs efficiently at a temperature at which the polymerase enzyme will be sufficiently active as not to require such a temperature shift; whether or not such a shift is needed in a given circumstance will be apparent to the one of ordinary skill in the art depending upon the enzyme used, and can also be determined empirically without undue experimentation.
  • labeled nucleic acid probe and “labeled probe” are used interchangeably herein and refer to an oligonucleotide that includes a detectable label that is directly or indirectly detectable to provide a signal.
  • a labeled-nucleic acid probe can be, for example, between 9-100 nucleotides in length, between 9-95 nucleotides in length, between 9-90 nucleotides in length, between 9-85 nucleotides in length, between 9- 80 nucleotides in length, between 9-75 nucleotides in length, between 9-70 nucleotides in length, between 9-65 nucleotides in length, between 9-60 nucleotides in length, between 9-55 nucleotides in length, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-100 nucle
  • 13-80 nucleotides in length between 13-80 nucleotides in length, between 13-70 nucleotides in length, between 13-60 nucleotides in length, between 13-50 nucleotides in length, between
  • nucleotides in length between 13-30 nucleotides in length, between 13-20 nucleotides in length, between 14-100 nucleotides in length, between 14-90 nucleotides in length, between
  • 14-80 nucleotides in length between 14-70 nucleotides in length, between 14-60 nucleotides in length, between 14-50 nucleotides in length, between 14-40 nucleotides in length, between
  • nucleotides in length between 15-60 nucleotides in length, between 15-50 nucleotides in length, between 15-40 nucleotides in length, between 15-30 nucleotides in length, or between 15-20 nucleotides in length.
  • the labeled-nucleic acid probe can be, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
  • a labeled-nucleic acid probe can comprises one, two, three, four or more detectable labels. Further, a detectable label can be at the 3 ’-end, 5 ’-end or at an internal position of the labeled-nucleic acid probe.
  • probes labeled with distinguishable detectable labels can be used.
  • fluorescent compounds Many suitable forms of these fluorescent compounds are available and can be used. Methods for fluorophore labeling of nucleic acids are known in the art. For multiplex approaches, probes labeled with fluorophores with distinguishable excitation and/or emission spectra can be used.
  • detectable labels include luminescent and bioluminescent markers (e.g., biotin, luciferase (e.g.., bacterial, firefly, click beetle and the like), luciferin, and aequorin), radiolabels (e.g.., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g.., galactosidases, glucorinidases, phosphatases (e.g.., alkaline phosphatase), peroxidases (e.g.., horseradish peroxidase), and cholinesterases), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g.., polystyrene, polypropylene, and latex) beads.
  • luminescent and bioluminescent markers e.g., biotin, luciferase (e.g.., bacterial, firefly, click beetle
  • a “biomolecule” is any molecule that is produced by a living organism, including large macromolecules such as proteins (e.g., antibodies), nucleic acids (e.g., DNA and RNA such as mRNA), polysaccharides, lipids and as well as small molecules such as primary metabolites, secondary metabolites, and natural products.
  • the target is a protein, e.g., an antibody.
  • the target is RNA, e.g., mRNA.
  • the target is DNA.
  • the target is a small molecule.
  • the target is a protein such as, for example, proteins of a cellular environment (e.g., intracellular or membrane proteins).
  • proteins include, without limitation, fibrous proteins such as cytoskeletal proteins (e.g., actin, arp2/3, coronin, dystrophin, FtsZ, keratin, myosin, nebulin, spectrin, tau, titin, tropomyosin, tubulin and collagen) and extracellular matrix proteins (e.g., collagen, elastin, f-spondin, pikachurin, and fibronectin); globular proteins such as plasma proteins (e.g., serum amyloid P component and serum albumin), coagulation factors (e.g., complement proteins, Cl -inhibitor and C3- convertase, Factor VIII, Factor XIII, fibrin, Protein C, Protein S, Protein Z, Protein Z-related protease inhibitor, thrombin, Von Willebrand
  • fibrous proteins such as
  • targets include, without limitation, DNA, RNA, cDNA, or the DNA product of RNA subjected to reverse transcription, A23187 (Calcimycin, Calcium Ionophore), Abamectine, Abietic acid, Acetic acid, Acetylcholine, Actin, Actinomycin D, Adenosine, Adenosine diphosphate (ADP), Adenosine monophosphate (AMP), Adenosine triphosphate (ATP), Adenylate cyclase, Adonitol, Adrenaline, epinephrine, Adrenocorticotropic hormone (ACTH), Aequorin, Aflatoxin, Agar, Alamethicin, Alanine, Albumins, Aldosterone, Aleurone, Alphaamanitin, Allantoin, Allethrin, a-Amanatin, Amino acid, Amylase, Anabolic steroid, Anethole, Angiotensinogen, Aniso
  • A23187 Cal
  • the sample is derived from a human, animal or plant.
  • the biological sample is a tissue sample, preferably an organ tissue sample.
  • samples are human.
  • the sample can be obtained, for example, from autopsy, biopsy, muscle punch, or from surgery. It can be a solid tissue or solid tumor such as parenchyme, connective or fatty tissue, heart or skeletal muscle, smooth muscle, skin, brain, nerve, kidney, liver, spleen, breast, carcinoma (e.g., bowel, nasopharynx, breast, lung, stomach etc.), cartilage, lymphoma, meningioma, placenta, prostate, thymus, tonsil, umbilical cord or uterus.
  • the tissue can be a tumor (benign or malignant), cancerous or precancerous tissue.
  • the sample can be obtained from an animal or human subject affected by disease or other pathology or suspected of same (normal or diseased), or considered normal or healthy.
  • the sample is a cell, e.g., a biological cell.
  • the sample is a tissue sample.
  • Samples including cell and/or tissue samples, can be fixed to avoid deterioration over subsequent staining and detection steps.
  • the sample can be fixed as soon after collection as possible.
  • fixatives include, but are not limited to, paraformaldehyde (PF A) at various concentrations (commonly between 1% and 5%, e.g., 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2,4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8% or 5.0%), formaldehyde at various concentrations (e.g., 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% formaldehyde), 10% neutral buffered formalin, Bouin’s solution, methanol, acetone, glutaraldehyde, etc.
  • PF A paraformaldehyde
  • formaldehyde at various concentrations (e.g.,
  • a biological samples e.g., cell and/or tissue samples
  • the fixative best suited for the cell or tissue sample and technique to be performed.
  • the fixative that can be used is 4% paraformaldehyde.
  • the sample e.g., cell and/or tissue sample
  • the sample can be embedded in a medium facilitating, for example, sectioning for histology and/or imaging. Paraffin- embedding of fixed samples is well known in the art.
  • a sample e.g., a tissue sample
  • pre-embedding of paraffin involves dehydration of tissues in increasing concentrations of alcohol, and then gradual replacement of alcohol by a paraffin solvent.
  • a paraffin solvent include xylene.
  • the sample is then embedded with melted paraffin using a mold, and hardened.
  • paraffin wax include, but are not limited to epoxy, acrylic, agar, gelatin, and celloidin.
  • a paraffin-embedded tissue sample undergoes sectioning, wherein the sample is cut into thin slices, or sections, to be placed on a slide. These sections can generally be around 5pm thick, though they can be thinner or thicker depending upon tissue type, target molecule, and label/label detection used, among other parameters.
  • sectioning After the sections are cut, they are transferred to a warm water bath and placed on a charged slide. Slides are dried, allowing for the removal of excess wax.
  • the sample can undergo staining.
  • Stains provide contrast to sections of sample, making viewing structures of the sample easier.
  • Exemplary stains include, but are not limited to alcian blue, aldehyde fuchsin, alkaline phosphatase, Bielshowsky stain, Congo red, crystal violet, eosin, Fontana- Masson, Giesmsa, Haematoxylin, Luna stain, Nissl, Period Acid Schiff (PAS), Red Oil 3, Reticulin stain, Sudan black, toluidine blue, and van Gieson.
  • PES Period Acid Schiff
  • staining is also used in reference to the detection of particular target molecules, e.g., using target-binding ligands or molecules as described herein applied to cell or tissue samples. Additional details on histology can be found, for example, in Ross, M. H. et al. Histology: a text and atlas with correlated cell and molecular biology (7th ed.) Wolters Kluwer. (ISBN: 978-1451187427).
  • Embodiments of the various aspects described herein include digestion or cleavage with a nuclease.
  • the nuclease can be an endonuclease or exonuclease.
  • the nuclease can be an RNA specific or DNA specific nuclease.
  • the nucleases used can include naturally occurring nucleases as well variants thereof and modified nucleases.
  • the nuclease is an DNase.
  • the DNase can be selected from eukaryotic DNases (e.g., mammalian DNases, bacterial DNases, or fungal DNases) or prokaryotic DNases.
  • Exemplary DNases include, but are not limited to DNase I, DNase II, DNase III, DNase IV, DNase V, DNase VI, and DNase VII.
  • the DNase is DNase I.
  • kinetics of digestion or cleavage by a nuclease can be controlled by varying temperature, time, and buffer/salt conditions, for example.
  • Nuclease like most enzymes, are sensitive to many buffer conditions, including ionic strength, pH and types of metal ions present (e.g., sodium ions vs. magnesium ions).
  • the temperature at which digestion or cleavage by the nuclease is performed can vary from, for example, 4°C to 65°C (e.g., 4°C, 25°C, 37°C, 42°C or 65°C).
  • the temperature at which the step of digestion or cleavage by the nuclease is performed is at about 4-25°C, 4-30°C, 4-35°C, 4-40°C, 4-45°C, 4-50°C, 4-55°C, 4-60°C, 10-25°C, 10-30°C, 10- 35°C, 10-40°C, 10-45°C, 10-50°C, 10-55°C, 10-60°C, 25-30°C, 25-35°C, 25-40°C, 25-45°C, 25-50°C, 25-55°C, 25-60°C, 25-65°C, 35-40°C, 35-45°C, 35-50°C, 35-55°C, 35-60°C, or 35- 65°C.
  • the step of digestion or cleavage by the nuclease is performed at about room temperature. In some other embodiments, the step of digestion or cleavage by the nuclease is performed at about 37°C.
  • the step of digestion or cleavage by the nuclease can be performed for about 30 minutes to about 24 hours. In some embodiments of any of the aspects, the step of digestion or cleavage by the nuclease step can be carried out for about 10 min, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours or about 24 hours.
  • Exemplary buffer formulations for digestion or cleavage by a nuclease include but are not limited to: Thermopol, NEB buffers 1,2, 3, 4, CutSmart, or customs buffers made with 0.5 to 2X PBS or 5 to 200 mM Tris-HCl or 5-200 mM Potassium Acetate or 5-200 mM Magnesium Acetate, 5-200 mM Tris-Acetate or 5-200mM bis-tris-propance HCL.
  • the buffers can be used with one or more of the following additives to modulate enzyme activity: 1-50 mM KC1, 1-20 mM MgSO4, 1-20 mM MgC12, 1-5 mM DTT, 0-500 pg/ml BSA, 1-500 NaCl, 0.01% to 0.5% Triton X-100 at pH values of 6-9.5.
  • the method comprises a step of extending the oligonucleotide primer, e.g., using a single-stranded extender template with a polymerase.
  • Method of synthesizing nucleic acid strand using polymerases are well known in the art and available to one of ordinary skill in the art. It is noted that kinetics of extension with a polymerase (i.e., polymerization) can be controlled by varying temperature, time, buffer/salt conditions, and deoxyribonucleotide triphosphate (dNTP) concentrations, for example.
  • dNTP deoxyribonucleotide triphosphate
  • Polymerases like most enzymes, are sensitive to many buffer conditions, including ionic strength, pH and types of metal ions present (e.g., sodium ions vs. magnesium ions).
  • the temperature at which polymerization step is performed can vary from, for example, 4°C to 65°C (e.g., 4°C, 25°C, 37°C, 42°C or 65°C).
  • the temperature at which the polymerization step is performed is about 4-25°C, 4- 30°C, 4-35°C, 4-40°C, 4-45°C, 4-50°C, 4-55°C, 4-60°C, 10-25C, 10-30°C, 10-35°C, 10-40°C, 10-45°C, 10-50°C, 10-55°C, 10-60°C, 25-30°C, 25-35°C, 25-40°C, 25-45°C, 25-50°C, 25- 55°C, 25-60°C, 25-65°C, 35-40°C, 35-45°C, 35-50°C, 35-55°C, 35-60°C, or 35-65°C.
  • the polymerization step is performed at about room temperature. In some other embodiments, the polymerization step is performed at about 37 °C. [00134] An extension or polymerization step can be performed (incubated) for about 30 minutes to about 24 hours.
  • the extension or polymerization step can be carried out for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours or about 24 hours.
  • the polymerase used to extend oligonucleotide primers and generate concatemers can be any of a number of template-dependent nucleic acid polymerases.
  • the polymerase is thermostable, such that it can withstand heating to a temperature and for a time sufficient to denature or dissociate double-stranded nucleic acids, retaining template-dependent polymerization activity when the reaction mixture is cooled to a temperature permitting annealing of extender template(s) to oligonucleotide primer(s) and primer extension.
  • Different thermostable polymerases have different reaction buffer and extension temperature optima; these parameters are known to those of ordinary skill in the art and/or described in product literature for given polymerases.
  • thermostable polymerases useful in the methods, compositions and kits described herein include the following.
  • polymerases that can be used in the methods described herein include but are not limited to: Standard Taq DNA polymerase (Cat. No. 10342053, Invitrogen, Carlsbad, CA), Platinum II Taq Hot-Start DNA Polymerase (Cat. No. 14966001, Invitrogen, Carlsbad, CA), Platinum SuperFi II DNA Polymerase (Cat. No. 12361010, Invitrogen, Carlsbad, CA), USBTM CycleSeqTM Thermostable DNA Polymerase (Cat. No. 792001000UN, Applied Biosystems, Waltham, MA); Taq DNA Polymerase (Cat. No. No.
  • the polymerase is a DNA polymerase such as a DNA polymerase having DNA strand displacement activity (a strand displacing polymerase).
  • DNA polymerase having DNA strand displacement activity a DNA polymerase having DNA strand displacement activity
  • “Strand displacement” describes the ability to displace downstream DNA encountered during synthesis.
  • Examples of polymerases having DNA strand displacement activity include, without limitation, phi29 DNA polymerase (e.g., NEB #M0269), Bst DNA polymerase, large fragment (e.g., NEB #M0275), or Bsu DNA polymerase, large fragment (e.g., NEB #M0330).
  • Other polymerases having strand displacement activity can be used.
  • Exemplary buffers that can be used for the extension step include but are not limited to: Thermopol® buffer; NEB® buffers 1,2, 3, 4; CutSmart® buffer; Isothermal Amplification Buffer; and the like.
  • Custom buffers can be made with 0.5 to 2X PBS; 5 to 200 mM Tris-HCl; 5-200 mM Potassium Acetate; 5-200 mM Magnesium Acetate; 5-200 mM Tris- Acetate; or 5- 200 mM Bis-Tris-Propane-HCl can be used with the addition one or all of these additives to modulate the enzyme activity (e.g., 1-50 mM KC1, 1-20 mM MgSO4, 1-20 mM MgC12, 1-5 mM DTT, 0-500 ug/ml BSA, 1-500 NaCl, 0.01% to 0.5% Triton X-100 at pH values of 6-9.5).
  • Custom buffers can be made with 0.5 to 2X PBS; 5 to 200
  • the buffer can also include dNTPs (e.g., dATP, dCTP, dGTP and dTTP).
  • dNTPs e.g., dATP, dCTP, dGTP and dTTP.
  • the omitted nucleotides serve as stoppers for the polymerase action, optionally with functional modifications.
  • kits for detecting, quantifying, and/or imaging target molecules comprises at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.
  • the components e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.
  • the kit comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.
  • the kit comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.
  • the kit comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; (iii) a nuclease; and (iv) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.
  • the kit comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease, optionally, the kit further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and/or buffers.
  • dNTPs deoxyribonucleotide triphosphates
  • the kit comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, optionally, the kit further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and/or buffers.
  • dNTPs deoxyribonucleotide triphosphate
  • the kit described herein lend itself well to multiplex labeling, detection and/or imaging. Accordingly, in some embodiments, the kit comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nucleas
  • the kit comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to
  • the kit comprises: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a
  • the kit comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable
  • the kit further comprises means for detecting the detectable label, e.g., a signal produced by the detectable label.
  • the kit further comprises instructions for use.
  • the system comprises at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.
  • the components e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.
  • the system comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.
  • the system comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; (iii) a nuclease; and (iv) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.
  • the system comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease, optionally, the system further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and/or buffers.
  • dNTPs deoxyribonucleotide triphosphates
  • the system comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, optionally, the system further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and/or buffers.
  • dNTPs deoxyribonucleotide triphosphate
  • the system comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nucleas
  • the system comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to
  • the system comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable
  • the system further comprises one or more reagents for digestion or cleavage by a nuclease such salts and/or buffers.
  • the system further comprises means for detecting the detectable label, e.g., a signal produced by the detectable label.
  • the system further comprises means for detecting the detectable label.
  • the reaction mixture comprises at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.
  • the components e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.
  • the reaction mixture comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.
  • the reaction mixture comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.
  • the reaction mixture comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; (iii) a nuclease; and (iv) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.
  • the composition comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease.
  • the first detectable label and the second detectable label can be distinguishable from each other.
  • the composition comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary
  • the composition comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable
  • the composition further comprises one or more reagents for digestion or cleavage by a nuclease such salts and/or buffers.
  • Nucleic acid molecules e.g., nucleic acid tags, labeled-nucleic acid probes, blocking probes, oligonucleotide primers, and single-stranded extender templates, described herein can comprise one or more nucleic acid modifications.
  • Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, inter-sugar linkage modifications, conjugates e.g., ligands), and any combinations thereof.
  • 6-(methyl)adenine 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine.
  • N 6 N 6 -(dimethyl)adenine, 2-(alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8- (hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2- (thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5- (alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine
  • alkylpseudouracil 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2- (thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)- 2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1 -substituted pseudouracil, 1 -substituted 2(thio)-pseudouracil, 1 -substituted 4-(thio)pseudouracil, 1 -substituted 2,4- (dithio)pseudouracil, 1 -(aminocarbonylethylenyl
  • Exemplary sugar modifications include, but are not limited to, 2’ -Fluoro, 3 ’-Fluoro, 2’- OMe, 3’-0Me, 2’ -deoxy modifications, and acyclic nucleotides, e.g., peptide nucleic acids (PNA), unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).
  • PNA peptide nucleic acids
  • UNA unlocked nucleic acids
  • GNA glycol nucleic acid
  • a nucleic acid modification can include replacement or modification of an inter-sugar linkage.
  • the nucleic acid tag comprises a nucleic acid modification capable of enhancing binding or hybridizing with a blocking probe.
  • Embodiment 3 The method of Embodiment 2, wherein the step of contacting the first nucleic acid tags with the first set of distinguishably-labeled nucleic acid probes comprises contacting the first nucleic acid tags with a set of probe, wherein the first set of probes comprises a first set of blocking-nucleic acid probes and the first set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein a concentration of the blocking-nucleic acid probes in the first set of blocking-nucleic acid probes is lower than a concentration of the labeled nucleic acid probes in the first set of blocking-nucleic acid probes.
  • Embodiment 4 The method of Embodiment 2, wherein the step of contacting the first nucleic acid tags with the first set of distinguishably-labeled nucleic acid probes comprises contacting the first nucleic acid tags with a set of probe, wherein the first set of probes comprises a first set of blocking-nucleic acid probes and the first set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag having a sequence complementary to the distinguishably-labeled nucleic acid probes.
  • Embodiment 8 The method of any one of Embodiments 1-6, wherein the first and second target molecules are same.
  • Embodiment 9 The method of any one of Embodiments 1-8, wherein the first or second target-binding ligand comprises a protein.
  • Embodiment 10 The method of any one of Embodiments 1-9, wherein the first or second target-binding ligand comprises an antibody or antigen-binding fragment thereof.
  • Embodiment 11 The method of any one of Embodiments 1-8, wherein the first or second target-binding ligand comprises RNA.
  • Embodiment 13 The method of any one of Embodiments 1-12, wherein the nuclease is a DNase.
  • Embodiment 74 A system comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe

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Abstract

La divulgation concerne des procédés, une composition, des kits et des systèmes pour détecter, quantifier et/ou imager des molécules cibles, par exemple, des biomolécules, dans un échantillon tel qu'une préparation de cellules ou de tissus.
PCT/US2025/012034 2024-01-19 2025-01-17 Procédés, kits, systèmes et compositions pour la détection multiplexe de cibles Pending WO2025155821A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230031305A1 (en) * 2021-07-30 2023-02-02 10X Genomics, Inc. Compositions and methods for analysis using nucleic acid probes and blocking sequences
US20230203567A1 (en) * 2020-04-22 2023-06-29 President And Fellows Of Harvard College Isothermal methods, compositions, kits, and systems for detecting nucleic acids

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230203567A1 (en) * 2020-04-22 2023-06-29 President And Fellows Of Harvard College Isothermal methods, compositions, kits, and systems for detecting nucleic acids
US20230031305A1 (en) * 2021-07-30 2023-02-02 10X Genomics, Inc. Compositions and methods for analysis using nucleic acid probes and blocking sequences

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