Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • 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
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
• • 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
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

• conventional, heterogeneous, hybridization assays typically comprise the following steps: immobilization of a target nucleic acid (e.g., on paper, beads, or plastic surfaces); addition of labelled probes that are complementary to the sequence of the target; hybridization; removal of unhybridized probes; and detection of the probes remaining bound to the immobilized target.
• a target nucleic acid e.g., on paper, beads, or plastic surfaces
• addition of labelled probes that are complementary to the sequence of the target e.g., on paper, beads, or plastic surfaces
• detection of the probes remaining bound to the immobilized target e.g., using solid surfaces to immobilize the target nucleic acids lengthens the time it takes for hybridization by restricting the mobility of, or access to, the target by the probes.
• solid surfaces may interfere with signal from the probes or lead to noise in the signal.
• M is, at each occurrence, independently the same or different chromophore;
• L 1a is, at each occurrence, independently a heteroarylene linker;
• L 2 and L 8 are independently optional linkers;
• L 1b , L 3 , L 5 , L 6 and L 7 are, at each occurrence, independently optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linkers;
• L 4 is, at each occurrence, independently an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linker;
• composition comprising: (1) a capture probe comprising: (i) a first segment comprising a first polynucleotide having a first sequence; and (ii) a second segment conjugated to the first segment, the second segment comprising a plurality of alternating second polynucleotides and first polymers, each of the first polymers comprising a first chromophore, each of the second polynucleotides comprising a second sequence; and (2) a plurality of detectable probes, each of the plurality of detectable probes comprising a third polynucleotide covalently bound to at least one a second polymer comprising a second chromophore, the third polynucleotide having a third sequence that has at least 90% complementarity to the second sequence.
• alkylene chain refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one carbon-carbon double bond and having from two to twelve carbon atoms, e.g., ethenylene, propenylene, n butenylene, and the like.
• the alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond.
• Alkylethers include at least one carbon oxygen bond, but may include more than one.
• polyethylene glycol PEG
• an alkylether group is optionally substituted.
• Alkoxy refers to a group of the formula ⁇ OR a where R a is an alkyl group as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group is optionally substituted.
• Heteroalkyl refers to an alkyl group, as defined above, comprising at least one heteroatom (e.g., N, O, P, or S) within the alkyl group or at a terminus of the alkyl group.
• the heteroatom is within the alkyl group (i.e., the heteroalkyl comprises at least one carbon-[heteroatom]x-carbon bond, where x is 1, 2 or 3).
• the heteroatom is at a terminus of the alkyl group and thus serves to join the alkyl group to the remainder of the molecule (e.g., M 1 -H-A), where M 1 is a portion of the molecule, H is a heteroatom, and A is an alkyl group).
• a heteroalkyl group is optionally substituted.
• exemplary heteroalkyl groups include ethylene oxide (e.g., polyethylene oxide), optionally including phosphorous-oxygen bonds, such as phosphodiester bonds.
• “Heteroalkoxy” refers to a group of the formula ⁇ OR a where R a is a heteroalkyl group as defined above containing one to twelve carbon atoms.
• n 25.
• Multimers may comprise, for example, the following structure: wherein x is 0 or an integer greater than 0, for example, x ranges from 0-100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
• “Heteroalkenylene” is a heteroalkylene, as defined above, comprising at least one carbon-carbon double bond. Unless stated otherwise specifically in the specification, a heteroalkenylene group is optionally substituted.
• Heteroalkynylene is a heteroalkylene comprising at least one carbon-carbon triple bond. Unless stated otherwise specifically in the specification, a heteroalkynylene group is optionally substituted.
• Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decalinyl, 7,7 dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless stated otherwise specifically in the specification, a cycloalkyl group is optionally substituted.
• Aryl refers to a ring system comprising at least one carbocyclic aromatic ring. In some embodiments, an aryl comprises from 6 to 18 carbon atoms. The aryl ring may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
• FRET refers to Förster resonance energy transfer refers to a physical interaction whereby energy from the excitation of one moiety (e.g., a first chromophore or “donor”) is transferred to an adjacent moiety (e.g., a second chromophore or “acceptor”). “FRET” is sometimes also used interchangeably with fluorescence resonance energy transfer (i.e., when each chromophore is a fluorescent moiety).
• a donor and acceptor that have these similar resonance frequencies are referred to as a “donor-acceptor pair(s),” which is used interchangeably with “FRET moieties” or “FRET dyes.”
• Acceptor or “acceptor chromophore” refers to a chromophore (e.g., a fluorophore) to which excitation energy from a donor chromophore is transferred via a non-radiative transfer through long-range dipole-dipole interaction.
• “Stoke’s shift” refers to a difference between positions (e.g., wavelengths) of the band maxima of absorption and emission spectra of an electronic transition (e.g., from excited state to non-excited state, or vice versa).
• isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 36Cl, 123I, and 125I, respectively.
• Isotopically-labeled compounds of Structure (I) or (II) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described below and in the following Examples using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
• Salts derived from organic bases include, for example, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2 dimethylaminoethanol, 2 diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N ethylpiperidine, polyamine resins and the like.
• basic ion exchange resins such as ammonia,
• the compounds of the present invention may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms.
• the compounds of the invention may be true solvates, while in other cases the compounds of the invention may merely retain adventitious water or another solvent or be a mixture of water plus some adventitious solvent.
• Probes 305a, 305b, 305c, 305d are independently compounds of structure (I). Each of the probes independently comprises a polymer 314a, 314b, 314c, 314d arranged between a second polynucleotide 316a, 316b, 316c, 316d and a third polynucleotide 317a, 317b, 317c, 317d. As shown in FIG. 3A, the second polynucleotide 316b of probe 305b hybridizes to the third polynucleotide 317a of probe 305a. In some embodiments, a triplex structure is formed by the polynucleotides of three probes.
• M is the same donor fluorophores at each occurrence. In other embodiments, M is the same acceptor fluorophore at each occurrence.
• compounds of the present disclosure have the following structure (IIa) or (IIb): or a stereoisomer, salt or tautomer thereof, wherein: M 1 is, at each occurrence, independently the same or different donor chromophores; M 2 is, at each occurrence, independently the same or different acceptor chromophores; L 1a is, at each occurrence, independently a heteroarylene linker; L 2 and L 8 are independently optional linkers: L 1b , L 3 , L 5 , L 6 , and L 7 are, at each occurrence, independently optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linkers; L 4 is, at each occurrence, independently an alkylene, alkenylene, alkynylene, heteroalkylene, hetero
• L′ is a heteroalkylene linker to a solid support, a solid support residue or a nucleoside.
• L′ comprises an alkylene oxide or phosphodiester moiety, or combinations thereof.
• At least one occurrence of L 4 is alkylene, or wherein each occurrence of L 4 is alkylene.
• at least one alkylene is ethylene, or wherein each alkylene is ethylene.
• at least one occurrence of R 3 is H, or wherein each occurrence of R 3 is H.
• L 1a is, at each occurrence independently an optionally substituted 5-7 membered heteroarylene linker.
• L 1a has one of the following structures: In embodiments, at least one occurrence of L 3 is an alkylene linker, or wherein each occurrence of L 3 is an alkylene linker.
• At least one occurrence of L 2 and/or L 8 is absent, or wherein each occurrence of L 2 and/or L 8 is absent.
• at least one occurrence of L 5 and/or L 6 is alkylene, or wherein each occurrence of L 5 and/or L 6 is alkylene.
• L 1b at each occurrence, independently comprises an amide functional group or a triazolyl functional group.
• R 5 is, at each occurrence, independently OH, O- or OR d .
• R 4 is, at each occurrence, oxo.
• the fluorophore is, at each occurrence, independently a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his- fluorophenyl-BODIPY, acridine, terrylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl- difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9, 10-ethynylanthracene, or ter- naphthyl moiety.
• the efficiency of the FRET process depends, in part, on characteristics of the chromophores. Specifically, high efficiency FRET requires a large overlap between the absorbance spectrum of the donor chromophore and the emission spectrum of the acceptor chromophore. Additionally, the distance and orientation of the chromophores plays an important role. FRET efficiency is inversely proportional to the 6 th power of the distance between the chromophores and the angle of the transition dipole moment should substantially align to be parallel (i.e., be near to 0° or 180°).
• the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 125° to 180°, from 130° to 180°, from 140° to 180°, from 150° to 180°, from 160° to 180°, from 170° to 180°, from 172° to 180°, from 175° to 180°, or from 177° to 180°. In certain embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 0° to 60°.
• the rings are fused.
• M 1 or M 2 or both at each occurrence, independently comprise three or more fused rings, four or more fused rings, five or more fused rings, or even six or more fused rings.
• M 1 or M 2 or both are cyclic.
• M 1 or M 2 or both are carbocyclic.
• M 1 or M 2 or both are heterocyclic.
• M 1 or M 2 or both at each occurrence, independently comprise an aryl moiety. In some of these embodiments, the aryl moiety is multicyclic.
• the substituent is a fluoro, chloro, bromo, iodo, amino, alkylamino, arylamino, hydroxy, sulfhydryl, alkoxy, aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, t-butyl, carboxy, sulfonate, amide, or formyl group.
• M 1 or M 2 or both are, at each occurrence, independently p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, or perylene amide, or a derivative thereof.
• M 1 or M 2 or both are, at each occurrence, independently a coumarin dye, resorufin dye, dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, energy transfer dye, thiazole orange dye, polymethine, or N-aryl-1,8-naphthalimide dye.
• M 1 and M 2 are, at each occurrence, independently boron-dipyrromethene, rhodamine, cyanine, pyrene, perylene, perylene monoimide, or 6-FAM or a derivative thereof. In still more embodiments of any of the foregoing, M 1 at each occurrence is the same.
• each M 1 is different. In still more embodiments, one or more M 1 is the same and one or more M 1 is different. In still more embodiments of any of the foregoing, M 2 at each occurrence is the same. In other embodiments, each M 2 is different. In still more embodiments, one or more M 2 is the same and one or more M 2 is different. In some embodiments, M 1 or M 2 or both are, at each occurrence, independently boron-dipyrromethene, rhodamine, cyanine, pyrene, perylene, perylene monoimide, or 6- FAM or a derivative thereof.
• each of the plurality of detectable probes comprising a third polynucleotide 116 covalently bound to at least one a second polymer comprising a second chromophore 118a, 118b.
• the third polynucleotide has a third sequence that has at least 90% complementarity to the second sequence.
• the second sequence has at least 92% complementarity to the third sequence. In some embodiments, the second sequence has at least 95% complementarity to the third sequence. In some embodiments, the second sequence has at least 96% complementarity to the third sequence. In some embodiments, the second sequence has at least 97% complementarity to the third sequence. In some embodiments, the second sequence has at least 98% complementarity to the third sequence. In some embodiments, the second sequence has at least 99% complementarity to the third sequence. In some embodiments, a triplex structure is formed by the polynucleotides of two detectable probes and the polynucleotide of the capture probe. As shown in FIG.
• each of the first polymers comprise a plurality of the first chromophores.
• each of the detectable probes comprise a plurality of the second chromophores.
• a second polymer 118a, 118b is covalently bound to each end of the third polynucleotide 116 of at least one of the detectable probes 106.
• the first chromophore is an acceptor chromophore.
• the first chromophore is a donor chromophore. As shown in FIG.
• the second chromophore is a donor chromophore. In other embodiments, the second chromophore is a donor chromophore.
• the third polynucleotide has a length ranging from 10 nucleotides to 40 nucleotides. In some embodiments, the third polynucleotide has a length ranging from 10 nucleotides to 30 nucleotides. In some embodiments, the third polynucleotide has a length ranging from 15 nucleotides to 25 nucleotides.
• each of the detectable probes have the structure (IIb) as described above.
• a fourth polynucleotide 120 covalently bound to a targeting moiety 122.
• a targeting moiety is covalently bound to the capture probe.
• a composition comprises a capture probe and a detectable probe as described herein.
• the third polynucleotide has a length ranging from 10 nucleotides to 40 nucleotides. In some embodiments, the third polynucleotide has a length ranging from 10 nucleotides to 30 nucleotides. In some embodiments, the third polynucleotide has a length ranging from 15 nucleotides to 25 nucleotides. In embodiments, the second sequence has at least 92% complementarity to the third sequence. In some embodiments, the second sequence has at least 95% complementarity to the third sequence. In some embodiments, the second sequence has at least 96% complementarity to the third sequence. In some embodiments, the second sequence has at least 97% complementarity to the third sequence.
• At least one of the detectable probes has the structure (I) as described above. In some embodiments, at least one of the detectable probes has the structure (II) as described above. In some embodiments, at least one of the detectable probes has the structure (IIa) as described above. In some embodiments, at least one of the detectable probes has the structure (IIb) as described above. In embodiments, each of the detectable probes have the structure (I), (II), (IIa), (IIb), or a combination thereof, described above. In some embodiments, each of the detectable probes have the structure (I) as described above (as illustrated in FIG. 2A).
• At least a portion of the detectable probes have the structure (I), (II), (IIa), (IIb), or a combination thereof, described above. In some embodiments, at least one of the detectable probes has the structure (I) as described above. In some embodiments, at least one of the detectable probes has the structure (II) as described above. In some embodiments, at least one of the detectable probes has the structure (IIa) as described above. In some embodiments, at least one of the detectable probes has the structure (IIb) as described above. In embodiments, each of the detectable probes have the structure (I), (II), (IIa), (IIb), or a combination thereof, described above.
• the capture probe 102 comprises (1) a first segment comprising a first polynucleotide having a first sequence, and (2) a second segment conjugated to the first segment, the second segment comprising a plurality of second polynucleotides 210 each comprising a second sequence. Also present are a plurality of branched linkers 226, and a plurality of detectable probes 204. Each of the plurality of detectable probes comprise a third polynucleotide 216 (having a third sequence) and each of the plurality of branched linkers comprise a fourth polynucleotide 228 (having a fourth sequence) and a fifth polynucleotide 224 (having a fifth sequence).
• a biotinylated target probe (5'-B-heg-ATGCACAGTCGG-dT-3') (SEQ ID NO: 1) bound to a neutravidin bead alone and mixed with (1) a first probe (5'-CGA CGC TTA CAG-heg-F-(heghegheg-F)3-heg-CCG ACT GTG CA-dT-3') (SEQ ID NO: 2) and a plurality of second probes (5'-CTGTAAGCGTCG-heg-F(heghegheg-F)9-heg- GACATTCGCAGC-dT-3') (SEQ ID NO: 3), (2) a first probe (5'-CGA CGC TTA CAG- heg-F-(heghegheg-F)3-heg-CCG ACT GTG CA-dT-3') (SEQ ID NO: 2), were analyzed using flow cytometry
• Probe stocks were heated using a thermal cycler to 94°C for 10 min, then cooled and maintained at 37°C until use.
• Five ng capture probe per sample was bound to neutravidin beads by incubation for 30 min at room temp in the dark. Beads were washed with hybridization buffer, centrifuged for 5 min at 400 xg and supernatant was removed. 200 pmol target amplifier was added and samples were incubated for 10 min at 37°C, followed by washing with hybridization buffer as described above. 400 pmole detection probe was added and samples were incubated for 10 min at 37°C. Samples were washed with hybridization buffer as described above and resuspended in cell staining buffer for analysis on the SA3800 flow cytometer.

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