Nucleotide probe

Abstract

To provide an improved probe achieving enhanced brightness and producing a further lowered signal-to-noise ratio.SOLUTION: A compound useful as a nucleic acid probe is disclosed. In some embodiments, the compound has the following structure (I), or is a stereoisomer, tautomer, or salt thereof. Methods relating to preparation and use of such a compound are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure is generally directed to compounds comprising a polymeric chromophore covalently attached to at least one polynucleotide (e.g., a compound comprising a polymeric fluorescent moiety attached to a nucleotide probe), as well as compositions and kits comprising same, and methods for their preparation and use in various analytical methods. [Background technology]

[0002] Nucleic acid hybridization probes are used to detect specific target sequences in a variety of diagnostic and analytical situations. Traditional, heterogeneous, hybridization assays typically involve the following steps: immobilization of the target nucleic acid (e.g., to paper, beads, or a plastic surface); addition of a labeled probe complementary to the target sequence; hybridization; removal of unhybridized probe; and detection of the probe that remains bound to the immobilized target. Unhybridized probes are removed by extensive washing of the hybridized target nucleic acid. This is generally the most time-consuming part of the procedure and often utilizes complex formats such as sandwich hybridization. Furthermore, the use of a solid surface to immobilize the target nucleic acid limits the mobility or accessibility of the target by the probe, thereby lengthening the hybridization time. Furthermore, the solid surface may interfere with or introduce noise into the signal from the probe. The conditions under which the probe-target hybrid is isolated also prevent in vivo detection and simultaneous detection of nucleic acids during synthesis reactions (real-time detection). Thus, there is a need in the art for improved probes with increased brightness and producing a lower signal-to-noise ratio. The present disclosure fulfills this need and further provides various related benefits. Summary of the Invention

[0003] Embodiments of the present application include compounds having the following structure (I) or a stereoisomer, salt, or tautomer thereof: [ka] (In the formula, M is, independently in each occurrence, either a) the same or different fluorophores, or b) the same or different fluorophore quenchers; L 1a is, independently in each occurrence, a heteroarylene linker; L 2 and L8 is independently an optional linker; L 1b , L 3 , L 5 , L 6 and L 7 is independently in each occurrence any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 is independently in each occurrence an alkylene, alkenylene, alkynylene, heteroaromatic group, an alkylene, heteroalkenylene, or heteroalkynylene linker; R 1 and R 2 each independently comprises a polynucleotide, or R 1 and R 2 comprises a polynucleotide, each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 3 is independently in each occurrence H, alkyl, or alkoxy; R 4 is independently in each occurrence OH, SH, O-, S-, OR d or SR d and R 5is independently in each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O-, S-, OR d or SR d and R c OH, SH, O - , S - , OR d , OL', SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, L' is, independently in each occurrence, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, or a linker comprising a covalent bond to a nucleoside; m, independently in each occurrence, is an integer greater than or equal to zero; n is an integer equal to or greater than 1, q and w are independently 0 or 1 in each occurrence if at least one occurrence of either q or w is 1.

[0004] In a further aspect, the application describes compositions comprising a compound of structure (Ia) and a compound of structure (Ib) or a stereoisomer, salt, or tautomer thereof. [ka] (In the formula, M 1 are, independently in each occurrence, the same or different fluorophores, M 2 are, independently in each occurrence, the same or different fluorophore quenchers, and L 1a is, independently in each occurrence, a heteroarylene linker; L2 and L 8 are independently an optional linker,

[0005] L 1b , L 3 , L 5 , L 6 and L 7 is independently in each occurrence any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 is independently in each occurrence an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; R 1a and R 2b each independently comprises a polynucleotide, or R 1a and R 2b comprises a polynucleotide, each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 1b and R 2a each independently comprises a polynucleotide, or R 1a and R 2b and if at least one of the groups comprises a polynucleotide, each independently is H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 3 is independently in each occurrence H, alkyl, or alkoxy;

[0006] R 4 is independently in each occurrence OH, SH, O-, S-, OR d or SR d and R 5is independently in each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O-, S-, OR d or SR d and R c OH, SH, O - , S - , OR d , OL', SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, L' is, independently in each occurrence, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, or a linker comprising a covalent bond to a nucleoside; m, independently in each occurrence, is an integer greater than or equal to zero; n is an integer equal to or greater than 1, q and w are independently 0 or 1 in each occurrence if at least one occurrence of either q or w is 1.

[0007] A further aspect of the present application includes a compound having the following structure (II) or a stereoisomer, salt, or tautomer thereof: [ka] (II) (In the formula, M 1 are, independently in each occurrence, the same or different fluorophores, M 2 is, independently in each occurrence, the same or different fluorophore-quencher;

[0008] L 1ais, independently in each occurrence, a heteroarylene linker; L 2 and L 8 are independently an optional linker, L 1b , L 3 , L 5 , L 6 and L 7 is independently in each occurrence any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 is independently in each occurrence an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 9 is a linker comprising a polynucleotide,

[0009] R 1a and R 2a each independently comprises a polynucleotide, R 1b and R 2b each independently comprise a polynucleotide or each independently comprise H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 3 is independently in each occurrence H, alkyl, or alkoxy; R 4 is independently in each occurrence OH, SH, O-, S-, OR d or SR d and R 5 is independently in each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O-, S-, OR d or SR d and

[0010] R c OH, SH, O - , S - , OR d , OL', SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, L' is, independently in each occurrence, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, or a linker comprising a covalent bond to a nucleoside; m, independently in each occurrence, is an integer greater than or equal to zero; n is an integer equal to or greater than 1, q and w are independently 0 or 1 in each occurrence if at least one occurrence of either q or w is 1.

[0011] Yet further embodiments of the present disclosure include compounds having the following structure (III) or a stereoisomer, salt, or tautomer thereof: [ka] (III) (In the formula, M 1 are, independently in each occurrence, the same or different fluorophores, M 2 is, independently in each occurrence, the same or different fluorophore-quencher;

[0012] L 1a is, independently in each occurrence, a heteroarylene linker; L 2 and L 8 are independently an optional linker, L 1b , L 3, L 5 , L 6 and L 7 is independently in each occurrence any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 is independently in each occurrence an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 9 is a linker comprising a polynucleotide, R 1a and R 2a each independently comprise a polynucleotide or each independently comprise H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and

[0013] R 1b and R 2b each independently comprises a polynucleotide, R 3 is independently in each occurrence H, alkyl, or alkoxy; R 4 is independently in each occurrence OH, SH, O-, S-, OR d or SR d and R 5 is independently in each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O-, S-, OR d or SR d and R c OH, SH, O - , S - , OR d , OL', SR d, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, L' is, independently in each occurrence, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, or a linker comprising a covalent bond to a nucleoside; m, independently in each occurrence, is an integer greater than or equal to zero; n is an integer equal to or greater than 1, q and w are independently 0 or 1 in each occurrence if at least one occurrence of either q or w is 1.

[0014] In yet a further aspect, the present disclosure provides a detectable probe for identifying the presence of a target nucleotide sequence, comprising: a first polynucleotide having a first end, the first polynucleotide having a first sequence comprising a target-complementary sequence having at least 90% complementarity to a target nucleotide sequence; a first polymer having a first end and a second end, the first end being covalently attached to the first end of the first polynucleotide, the first polymer comprising two fluorophores; and A detectable probe includes a second polynucleotide having a first end covalently attached to a second end of the first polymer.

[0015] In a further aspect, the present disclosure provides a detectable probe for identifying the presence of a target nucleotide sequence, comprising: a) a first polynucleotide covalently attached to a first polymer comprising two or more fluorophores; b) a second segment comprising a second nucleotide sequence covalently attached to a second polymer comprising two or more fluorophore quenchers; Including, the first nucleotide sequence comprises: i) a target-complementary sequence having at least 90% complementarity to a target nucleic acid sequence, capable of forming a double-stranded hybrid with the target sequence under assay conditions having a first strength; and ii) a probe-complementary sequence having at least 90% complementarity to at least a portion of a second nucleotide sequence, capable of forming a double-stranded hybrid with at least a portion of the second nucleotide sequence under assay conditions having a second strength, the second strength being less than the first strength; the first polymer, in the absence of the second polymer, when excited with ultraviolet light of a given wavelength, has a peak fluorescence emission that is at least 85% of the sum of the peak fluorescence emissions of the individual fluorophores present in the first polymer when excited with ultraviolet light of the same wavelength; It further comprises a detectable probe.

[0016] In a still further aspect, the present application includes kits comprising the compounds, compositions, or detectable probes described herein. In the figures, identical reference numbers identify similar elements. The sizes and relative positions of elements in the figures are not necessarily to scale, and some of these elements have been enlarged and positioned to improve legibility of the figures. Furthermore, the particular shapes of the elements as drawn are not intended to convey any information regarding the actual shape of the particular elements, but have been selected solely for ease of recognition of the figures. [Brief explanation of the drawings]

[0017] [Figure 1A] FIG. 1 shows a schematic diagram of a probe hybrid complex in which the first and second sequences are 100% complementary. [Figure 1B] FIG. 1 shows a schematic diagram of a probe hybrid complex in which the first and second sequences are 73% complementary. [Figure 1C] FIG. 1 shows a schematic diagram of a probe hybrid complex in which each pair of sequences is 100% complementary. [Figure 1D] FIG. 1 shows a schematic diagram of a probe according to some embodiments. [Figure 2] FIG. 1 depicts a melting curve plot of a particular embodiment comprising individual fluorescent probes and probe hybrid complexes, where the probes have complementary sequences and the quencher probe has a concentration of 6.4 μM. [Figure 3] FIG. 1 depicts a melting curve plot of a particular embodiment comprising individual fluorescent probes and a probe hybrid complex, where the probes have complementary sequences and the quencher construct has a concentration of 9.1 μM. [Figure 4] FIG. 1 shows melting curve plots of certain embodiments comprising individual fluorescent probes and probe hybrid complexes, where the probes have non-complementary sequences. [Figure 5] FIG. 10 plots quenching efficiency data of probe hybrid complexes as a function of temperature during melting curve analysis. [Figure 6] FIG. 1 depicts the relative fluorescence of individual fluorescent probe compounds containing increasing numbers of fluorescein moieties 1) as individual probes and 2) as hybrid complexes formed with complementary quencher constructs. [Figure 7] FIG. 1 depicts melting curve plots of certain embodiments comprising individual fluorescent probe compounds containing increasing numbers of fluorescein moieties 1) as individual probes and 2) as hybrid complexes formed with complementary quencher constructs. [Figure 8] FIG. 1 shows evidence of probe / quencher hybridization upon cooling using a thermal cycler. [Figure 9] FIG. 1 depicts the quenching efficiency observed for hybrid complexes compared to analogous variants. [Figure 10] FIG. 1 shows melting curve plots of certain embodiments including 1) individual fluorescent probe compounds and 2) hybrid complexes formed with complementary quencher constructs. [Figure 11]FIG. 1 illustrates the quenching efficiency of hybrid complexes formed from individual probes containing increasing numbers of fluorescent moieties and complementary quencher constructs with a single BHQ-1 moiety. [Figure 12] FIG. 1 illustrates the quenching efficiency of hybrid complexes containing one fluorescein moiety and one BHQ-1 moiety at various initial concentrations of the quencher construct. DETAILED DESCRIPTION OF THE INVENTION

[0018] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, it will be understood by those skilled in the art that the invention may be practiced without these details. Unless otherwise required by context, throughout this specification and claims, the word "comprise," and variations thereof, such as "comprises" and "comprising," are to be interpreted in an open and inclusive sense, i.e., "including, but not limited to." References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described with respect to that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0019] "Amino" refers to the group -NH2. "Carboxy" refers to the group -CO2H. "Cyano" refers to the radical -CN. "Formyl" refers to the group -C(=O)H. "Hydroxy" or "hydroxyl" refers to the group --OH. "Imino" refers to the group =NH. "Nitro" refers to the -NO2 group. "Oxo" refers to the =O substituent. "Sulfhydryl" refers to an -SH group. "Thioxo" refers to the group ═S. "Alkyl" refers to a straight or branched hydrocarbon chain radical, consisting solely of carbon and hydrogen atoms, containing no unsaturation, having 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl), attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1 methylethyl (isopropyl), n-butyl, n-pentyl, 1,1 dimethylethyl (t-butyl), 3 methylhexyl, 2 methylhexyl, etc. Unless specifically stated herein, alkyl groups may be substituted.

[0020] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen, containing no unsaturation, and having 1 to 12 carbon atoms, that connects the rest of the molecule to a radical group, e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated specifically herein, alkylene can be substituted. "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen, containing at least one carbon-carbon double bond, having 2 to 12 carbon atoms, that connects the rest of the molecule to a radical group, 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. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated specifically herein, alkenylene may be substituted.

[0021] "Alkynylene" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen, containing at least one carbon-carbon triple bond, having 2 to 12 carbon atoms, that connects the rest of the molecule to a radical group, e.g., ethenylene, propenylene, n-butenylene, and the like. The alkynylene 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. The points of attachment of the alkynylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated specifically herein, alkynylene may be optionally substituted. "Alkyl ether" refers to any alkyl group, as defined above, in which at least one carbon-carbon bond has been replaced with a carbon-oxygen bond. The carbon-oxygen bond may be terminal (as in an alkoxy group) or the carbon-oxygen bond may be internal (i.e., COC). An alkyl ether contains at least one carbon-oxygen bond, but may contain more than one. For example, polyethylene glycol (PEG) is included within the meaning of alkyl ether. Unless specifically stated herein, alkyl ether groups may be substituted. For example, in some embodiments, an alkyl ether is substituted with an alcohol or -OP(=Ra)(Rb)Rc, where each of Ra, Rb, and Rc is as defined for the compound of structure (I).

[0022] "Alkoxy" refers to a radical of the formula -ORa, where Ra is an alkyl radical as defined above containing 1 to 12 carbon atoms. Unless stated specifically in the specification, an alkoxy radical may be optionally substituted. "Alkoxyalkyl ether" refers to a group of formula -ORaRb, where Ra is an alkylene group as defined above containing 1 to 12 carbon atoms, and Rb is an alkyl ether group as defined herein. Unless specifically stated herein, an alkoxyalkyl ether may be substituted, for example, with an alcohol or -OP(=Ra)(Rb)Rc, where each of Ra, Rb, and Rc is as defined for compounds of structure (I).

[0023] "Heteroalkyl" refers to an alkyl group, as defined above, containing at least one heteroatom (e.g., N, O, P, or S) within the alkyl group or at a terminal end of the alkyl group. In some embodiments, the heteroatom is within the alkyl group (i.e., the heteroalkyl contains at least one carbon-[heteroatom]x-carbon bond, where x is 1, 2, or 3). In other embodiments, the heteroatom is at the terminal end of the alkyl group, thereby serving to attach the alkyl group to the remainder of the molecule (e.g., M1-HA, where M1 is a portion of the molecule, H is a heteroatom, and A is the alkyl group). Unless specifically stated herein, heteroalkyl groups may be substituted. Exemplary heteroalkyl groups include ethylene oxide (e.g., polyethylene oxide), which may contain a phosphorus-oxygen bond, e.g., a phosphodiester bond. "Heteroalkoxy" refers to a radical of the formula -ORa, where Ra is a heteroalkyl radical as defined above containing 1 to 12 carbon atoms. Unless stated specifically in the specification, a heteroalkoxy radical may be optionally substituted.

[0024] "Heteroalkylene" refers to an alkyl group, as defined above, containing at least one heteroatom (e.g., N, O, P, or S) within the alkylene chain or at a terminus of the alkylene chain. In some embodiments, the heteroatom is within the alkylene chain (i.e., the heteroalkylene contains at least one carbon-[heteroatom]-carbon bond, and x is 1, 2, or 3). In other embodiments, the heteroatom is at the terminus of the alkylene, thereby serving to attach the alkylene to the remainder of the molecule (e.g., M1-HA-M2, where M1 and M2 are part of the molecule, H is a heteroatom, and A is an alkylene group). Unless specifically stated otherwise in the specification, heteroalkylene groups may be substituted. Exemplary heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide), and the linking groups exemplified below: "C," "HEG," "TEG," "PEG 1K," and variations thereof. [ka] Included in various embodiments of heteroalkylene linkers are multimers of the C linker, HEG linker, and / or PEG 1K linker described above.

[0025] In some embodiments of the PEG 1K linker, n is 25. The multimer may, for example, comprise the following structure: [ka] (wherein x is an integer of 0 or greater than 0, for example, x is in the range of 0 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)) "Heteroalkenylene" is a heteroalkylene, as defined above, containing at least one carbon-carbon double bond. Unless stated specifically in the specification, a heteroalkenylene group may be optionally substituted.

[0026] A "heteroalkynylene" is a heteroalkylene containing at least one carbon-carbon triple bond. Unless stated specifically in the specification, a heteroalkynylene group may be optionally substituted. "Heteroatom," in reference to a "heteroatom linker," refers to a linker group consisting of one or more heteroatoms. Exemplary heteroatom linkers include single atoms selected from the group consisting of O, N, P, and S, as well as multiple heteroatoms, such as linkers having the formula -P(O-)(=O)O- or -OP(O-)(=O)O-, and multimers and combinations thereof. "Phosphate" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc, Rb is OH, O-, ORc, a thiophosphate group, or further phosphate group, and Rc is a counterion (e.g., Na+, etc.).

[0027] "Phosphoalkyl" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc, Rb is -Oalkyl, and Rc is a counterion (e.g., Na+, etc.). Unless specifically stated herein, a phosphoalkyl group may be substituted. For example, in certain embodiments, the -Oalkyl portion of a phosphoalkyl group may be substituted with one or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether, thiophosphoalkylether, or -OP(=Ra)(Rb)Rc, where each of Ra, Rb, and Rc is as defined for the compound of structure (I). "Phosphoalkyl ether" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc, Rb is an -Oalkyl ether, and Rc is a counterion (e.g., Na+, etc.). Unless specifically stated herein, a phosphoalkyl ether group may be substituted. For example, in certain embodiments, the -Oalkyl ether portion of a phosphoalkyl ether group may be substituted with one or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or -OP(=Ra)(Rb)Rc, where each of Ra, Rb, and Rc is as defined for the compound of structure (I).

[0028] "Thiophosphate" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S, Rb is OH, O-, S-, ORd, or SRd, Rc is OH, SH, O-, S-, ORd, SRd, a phosphate group, or an additional thiophosphate group, and Rd is a counterion (e.g., Na+), with the proviso that i) Ra is S, ii) Rb is S- or SRd, iii) Rc is SH, S-, or SRd, or iv) a combination of i), ii), and / or iii). "Thiophosphoalkyl" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S, Rb is OH, O-, S-, ORd, or SRd, Rc is -Oalkyl, and Rd is a counterion (e.g., Na+, etc.), with the proviso that i) Ra is S, ii) Rb is S- or SRd, or iii) Ra is S and Rb is S- or SRd. Unless specifically stated herein, a thiophosphoalkyl group may be substituted. For example, in certain embodiments, the -Oalkyl portion of a thiophosphoalkyl group may be substituted with one or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether, thiophosphoalkylether, or -OP(=Ra)(Rb)Rc, where each of Ra, Rb, and Rc is as defined for the compound of structure (I).

[0029] "Thiophosphoalkyl ether" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S, Rb is OH, O-, S-, ORd, or SRd, Rc is an -Oalkyl ether, and Rd is a counterion (e.g., Na+, etc.), with the proviso that i) Ra is S, ii) Rb is S- or SRd, or iii) Ra is S and Rb is S- or SRd. Unless specifically stated herein, a thiophosphoalkyl ether group may be substituted. For example, in certain embodiments, the -Oalkyl ether portion of a thiophosphoalkyl group may be substituted with one or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or -OP(=Ra)(Rb)Rc, where each of Ra, Rb, and Rc is as defined for the compound of structure (I).

[0030] "Carbocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing 3 to 18 carbon atoms. Unless specifically stated otherwise, a carbocyclic ring may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems and may be partially or fully saturated. Non-aromatic carbocyclyl radicals include cycloalkyl, while aromatic carbocyclyl radicals include aryl. Unless specifically stated otherwise, a carbocyclic group may be optionally substituted. "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, which may include fused or bridged ring systems, having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, saturated or unsaturated, and attached to the remainder of the molecule by a single bond. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decalinyl, 7,7 dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless specifically stated herein, cycloalkyl groups may be substituted.

[0031] "Aryl" refers to a ring system containing at least one carbocyclic aromatic ring. In some embodiments, an aryl contains 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. Aryl includes aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, and phenanthrene. Unless stated specifically in the specification, an aryl group may be optionally substituted.

[0032] "Heterocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing 1 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless specifically stated otherwise herein, a heterocycle may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; the nitrogen, carbon, or sulfur atoms of the heterocyclic ring may be oxidized; the nitrogen atom may be quaternized; and the heterocyclic ring may be partially or fully saturated. Examples of aromatic heterocyclic rings are listed below in the definition of heteroaryl (i.e., heteroaryl, which is a subset of heterocyclic). Examples of non-aromatic heterocyclic rings include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, and 1,1-dioxothiomorpholinyl. Unless specifically stated herein, heterocyclic groups may be substituted.

[0033] "Heteroaryl" refers to a 5- to 14-membered ring system containing 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For purposes of certain embodiments of the present invention, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems, and the nitrogen, carbon, or sulfur atom(s) of the heteroaryl radical may be optionally oxidized, and the nitrogen atom(s) may be optionally quaternized.Examples include azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzoindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4 benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzo Thienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2a]pyridinyl, benzoxazolinonyl, benzimidazolethionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl , naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, prilimdinonyl, pyri Heteroaryl groups include, but are not limited to, dazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl, quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated specifically in the specification, heteroaryl groups may be optionally substituted.

[0034] "Fused" refers to a ring system containing at least two rings, wherein the two rings share at least one common ring atom, for example, two common ring atoms. When the fused ring is a heterocyclyl ring or a heteroaryl ring, the common ring atom may be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tetracyclic, etc.

[0035] As used herein, the term "substituted" means that at least one hydrogen atom (e.g., one, two, three, or all hydrogen atoms) has been replaced with, for example, a halogen atom, e.g., F, Cl, Br, and I; an oxygen atom in groups such as a hydroxyl group, an alkoxy group, and an ester group; a sulfur atom in groups such as a thiol group, a thioalkyl group, a sulfone group, a sulfonyl group, and a sulfoxide group; a nitrogen atom in groups such as an amine, an amide, an alkylamine, a dialkylamine, an arylamine, an alkylarylamine, a diarylamine, an N-oxide, an imide, and an enamine; a trialkylsilyl group, a dialkylarylsilyl group, an alkyldi ... "Substituted" refers to any of the above groups (e.g., alkyl, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, alkoxy, alkyl ether, alkoxyalkyl ether, heteroalkyl, heteroalkoxy, phosphoalkyl, phosphoalkyl ether, thiophosphoalkyl, thiophosphoalkyl ether, carbocyclic, cycloalkyl, aryl, heterocyclic, and / or heteroaryl) replaced by a bond to a non-hydrogen atom, including, but not limited to, silicon in groups such as arylsilyl and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also refers to any of the above groups in which one or more hydrogen atoms are replaced by a higher bond (e.g., a double or triple bond) to a heteroatom, such as oxygen in oxo, carbonyl, carboxyl, and ester groups, and nitrogen in groups such as imine, oxime, hydrazone, and nitrile. For example, "substituted" includes any of the above groups in which one or more hydrogen atoms have been replaced with -NRgRh, -NRgC(=O)Rh, -NRgC(=O)NRgRh, -NRgC(=O)ORh, -NRgS0Rh, -OC(=O)NRgRh, -ORg, -SRg, -SORg, -S0Rg, -OS0Rg, -S00Rg, =NS0Rg, and -S0NRgRh."Substituted" also refers to any of the above groups in which one or more hydrogen atoms have been replaced with -C(=O)Rg, -C(=O)ORg, -C(=O)NRgRh, -CH2SO2Rg, -CH2SO2NRgRh, where Rg and Rh are the same or different and independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" further refers to any of the above groups in which one or more hydrogen atoms have been replaced by a bond to amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. In some embodiments, an optional substituent is -OP(=Ra)(Rb)Rc, where each of Ra, Rb, and Rc is as defined for compounds of structure (I). In addition, each of the foregoing substituents may also be substituted with one or more of the above-described substituents.

[0036] "Conjugation" refers to the overlap of one p orbital with another p orbital across an intervening sigma bond. Conjugation can occur in cyclic or acyclic compounds. "Degree of conjugation" refers to the overlap of at least one p orbital with another p orbital across an intervening sigma bond. For example, 1,3-butadiene has a degree of conjugation of 1, while benzene and other aromatic compounds typically have multiple degrees of conjugation. Fluorescent and colored compounds typically contain a degree of conjugation of at least 1. "Fluorescent" refers to a molecule that can absorb light of a particular frequency and emit light of a different frequency. Fluorescence is well known to those skilled in the art. "Colored" refers to molecules that absorb light within the color spectrum (i.e., red, yellow, blue, etc.).

[0037] "Linker" refers to a continuous chain of at least one atom, such as carbon, oxygen, nitrogen, sulfur, phosphorus, and combinations thereof, which connects one portion of a molecule to another portion of the same molecule, or to a different molecule, moiety, or solid support (e.g., a microparticle). A linker may connect molecules through covalent bonds or other means, such as ionic or hydrogen bonding interactions. As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" refers to either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and includes oligonucleotides.

[0038] A nucleic acid can represent a coding strand or its complement. A nucleic acid can be composed of monomers that are naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-mirror forms of naturally occurring nucleotides), or a combination of both. Nucleotides in a nucleic acid sequence are named according to standard IUPAC conventions. Specifically, "A" is adenine, "C" is cytosine, "G" is guanine, "T" is thymine, and "U" is uracil, which refer to the following structures: [ka]

[0039] The sequence of a polynucleotide refers to the order in which the nucleotides are arranged in the polynucleotide. Modified nucleotides can have modifications or substitutions in the sugar moiety or the pyrimidine or purine base moiety. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, and the like. In various embodiments, modified internucleotide bonds are used. Modified internucleotide bonds are well known in the art and include methylphosphonates, phosphorothioates, phosphorodithioates, phosphoramidites, and phosphate ester bonds. Nucleic acid molecules can be either single-stranded or double-stranded.

[0040] The terms "target," "target sequence," "target region," and "target nucleic acid," as used herein, refer to a region or subsequence of a nucleic acid to be detected. The term "hybridization," as used herein, refers to any method by which a first strand of nucleic acid binds to a second strand of nucleic acid through base pairing. Hybridization can occur between perfectly complementary nucleic acid strands or between "substantially complementary" nucleic acid strands that contain minor regions of mismatch. As used herein, a probe is a polynucleotide that is "specific" for a target sequence if, when used under sufficiently stringent conditions, the probe hybridizes primarily only to the target nucleic acid. Typically, a probe is specific for a target sequence if the probe-target duplex stability is greater than the stability of the duplex formed between the probe and any other sequence found in the sample. Those skilled in the art will recognize that various factors, such as salt conditions, as well as the base composition and mismatch position of the probe, affect probe specificity, and that routine experimentation to confirm probe specificity will be required in most cases. Hybridization conditions can be selected so that the probe can form a stable duplex only with the target sequence. Thus, the use of a target-specific probe under appropriately stringent conditions allows for the specific amplification of target sequences containing a target probe binding site. The use of sequence-specific conditions allows for the specific binding of the probe to target sequences containing an exactly complementary probe binding site.

[0041] Conditions under which only completely complementary nucleic acid strands hybridize are referred to as "stringent hybridization conditions" or "sequence-specific hybridization conditions." The term "stringent" as used herein refers to hybridization conditions that are generally understood in the art to define the conditions for hybridization processes. Stringent conditions can be low, high, or moderate, and these terms are generally known in the art and well-recognized by those skilled in the art. In various embodiments, stringent conditions can include, for example, high stringency conditions and / or moderate stringency (i.e., moderate stringency) conditions. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions, and the degree of mismatch tolerance can be controlled by appropriate adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability according to the guidelines provided by the art, empirically considering numerous variables, including, for example, the length and base pair composition of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs.

[0042] As used herein, "complementary" refers to a nucleic acid molecule that can form hydrogen bonds with another nucleic acid molecule, either by traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides. It is understood in the art that nucleic acid molecules do not need to be 100% complementary to the target nucleotide sequence that can be specifically hybridized. That is, two or more nucleic acid molecules do not need to be completely complementary, and this is indicated by the percentage of consecutive residues in the nucleic acid molecule that can form hydrogen bonds with the second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then 5 base pairs of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules represent 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively. "Perfectly" or "fully" complementary nucleic acid molecules mean that all contiguous residues of a first nucleic acid molecule will hydrogen bond with the same number of contiguous residues of a second nucleic acid molecule, where the nucleic acid molecules both have the same number of nucleotides (i.e., have the same length) or the two molecules have different lengths.

[0043] The term "hybridization complex," as used herein, refers to a complex formed between two nucleotide sequences due to the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases, which may be further stabilized by base-stacking interactions. Two complementary nucleotide sequences hydrogen bond in an antiparallel configuration. Hybridization complexes may be formed in solution (e.g., Cot or Rot analysis) or between one nucleotide sequence present in solution and another nucleotide sequence immobilized on a solid support (e.g., paper, membrane, filter, chip, pin, or glass slide, or any other suitable substrate on which cells and / or nucleic acids are immobilized). A "reactive group" is a moiety that can react with a second reactive group (e.g., a "complementary reactive group") to form one or more covalent bonds, for example, by substitution, oxidation-reduction, addition, or cycloaddition reactions. Exemplary reactive groups include, for example, nucleophiles, electrophiles, dienes, dienophiles, aldehydes, oximes, hydrazones, alkynes, amines, azides, acyl azides, acyl halides, nitriles, nitrones, sulfhydryls, disulfides, sulfonyl halides, isothiocyanates, imidoesters, activated esters, ketones, α,β-unsaturated carbonyls, alkenes, maleimides, α-haloimides, epoxides, aziridines, tetrazines, tetrazoles, phosphines, biotin, thiirane, and the like.

[0044] The terms "visible" and "visually detectable" are used herein to refer to substances observable by visual inspection without prior illumination or chemical or enzymatic activation. Such visually detectable substances absorb and emit light in the spectral region ranging from about 300 to about 900 nm. Preferably, such substances are deeply colored and have a molar extinction coefficient of at least about 40,000, more preferably at least about 50,000, even more preferably at least about 60,000, even more preferably at least about 70,000, and most preferably at least about 80,000 M cm. Compounds of the present invention can be observed and detected with the naked eye or with the aid of optically based detection devices, including, but not limited to, absorption spectrophotometers, transmission light microscopes, digital cameras, and scanners. Visually detectable substances are not limited to those that emit and / or absorb light within the visible spectrum. Also included within the scope of "visually detectable" substances are substances that emit and / or absorb light in the ultraviolet (UV) region (about 10 nm to about 400 nm), the infrared (IR) region (about 700 nm to about 1 mm), and substances that emit and / or absorb in other regions of the electromagnetic spectrum.

[0045] For purposes of embodiments of the present invention, the term "photostable visible dye" refers to a chemical moiety that is visually detectable as defined hereinabove and does not significantly change or decompose when exposed to light. Preferably, a photostable visible dye does not exhibit significant fading or decomposition after at least one hour of exposure to light. More preferably, the visible dye is stable after at least 12 hours, even more preferably at least 24 hours, even more preferably at least one week, and most preferably at least one month of exposure. Non-limiting examples of photostable visible dyes suitable for use in the compounds and methods of the present invention include azo dyes, thioindigo dyes, quinacridone pigments, dioxazines, phthalocyanines, perinones, diketopyrrolopyrroles, quinophthalones, and truarycarboniums. As used herein, the term "perylene derivative" is intended to include any substituted perylene that is visually detectable. However, the term is not intended to include perylene itself. The terms "anthracene derivative," "naphthalene derivative," and "pyrene derivative" are used analogously. In some preferred embodiments, the derivative (e.g., perylene, pyrene, anthracene, or naphthalene derivative) is an imide, bisimide, or hydrazamimide derivative of perylene, anthracene, naphthalene, or pyrene.

[0046] The visually detectable molecules of various embodiments of the present invention are useful in a wide variety of analytical applications, including biochemical and biomedical applications, in which the presence, location, or amount of a particular analyte (e.g., a biomolecule) needs to be determined. Accordingly, in another aspect, the present invention provides a method for visually detecting a biomolecule, comprising: (a) providing a biological system having a visually detectable biomolecule comprising a compound of Formula (I) linked to the biomolecule; and (b) detecting the biomolecule by its visual characteristics. For purposes of the present invention, the phrase "detecting a biomolecule by its visual characteristics" means that the biomolecule is observed without illumination or chemical or enzymatic activation, either with the naked eye or with the aid of an optically based detection device, including, but not limited to, an absorption spectrophotometer, a transmitted light microscope, a digital camera, and a scanner. Densitometers may be used to quantify the amount of visually detectable biomolecule present. For example, the relative amounts of biomolecules in two samples can be determined by measuring their relative optical densities. Knowing the theoretical amount of dye molecules per biomolecule and the extinction coefficient of the dye molecule, the absolute concentration of the biomolecule can also be determined from optical density measurements. As used herein, the term "biological system" refers to any solution or mixture containing one or more biomolecules in addition to visually detectable biomolecules. Examples of such biological systems include cells, cell extracts, tissue samples, electrophoresis gels, assay mixtures, and hybridization reaction mixtures.

[0047] "Solid support" refers to any solid substrate known in the art for solid phase support of molecules, for example, "microparticle" refers to any number of small particles useful for binding to the compounds of the invention, including, but not limited to, glass beads, magnetic beads, polymeric beads, non-polymeric beads, etc. In certain embodiments, the microparticle comprises a polystyrene bead. "Solid support residue" refers to a functional group that remains attached to a molecule when the molecule is cleaved from the solid support. Solid support residues are known in the art and can be readily derived based on the structure of the solid support and the group linking the molecule to it.

[0048] A "targeting moiety" is a moiety that selectively binds to or associates with a particular target, e.g., an analyte molecule. "Selectively" binding or associating means that the targeting moiety preferentially associates or binds to the desired target over other targets. In some embodiments, the compounds disclosed herein include binding to a targeting moiety for the purpose of selectively binding to or associating with a compound having the analyte of interest (i.e., the target of the targeting moiety), thereby enabling detection of the analyte. Exemplary targeting moieties include, but are not limited to, antibodies, antigens, nucleic acid sequences, enzymes, proteins, cell surface receptor antagonists, and the like. In some embodiments, the targeting moiety is a moiety, e.g., an antibody, that selectively binds to or associates with a target feature on a cell, e.g., a target feature on a cell membrane or other cellular structure, thereby enabling detection of the cell of interest. Small molecules that selectively bind to or associate with a desired analyte are also considered targeting moieties in certain embodiments. Those skilled in the art will understand that other analytes and corresponding targeting moieties are useful in various embodiments. "Base-pairing moiety" refers to a heterocyclic moiety that can hybridize with a complementary heterocyclic moiety through hydrogen bonding (e.g., Watson-Crick base pairing). Base-pairing moieties include natural and unnatural bases. Non-limiting examples of base-pairing moieties are RNA and DNA bases, such as adenosine, guanosine, thymidine, cytosine, and uridine, and their analogs.

[0049] The embodiments disclosed herein are also intended to encompass all compounds of structure (I), (II), or (III) that are isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, 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 methods analogous to those described below and in the Examples below, substituting the appropriate isotopically labeled reagent for the previously used non-labeled reagent. A "stable compound" or "stable structure" is intended to indicate a compound that is sufficiently rigid to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and that the description includes both substituted and unsubstituted alkyl groups.

[0050] "Salts" includes both acid and base addition salts. "Acid addition salts" include salts formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, citric acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, These refer to salts formed with glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0051] "Base addition salt" refers to a salt prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, salts of cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dianol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0052] Crystallization can produce solvates of the compounds described herein. Embodiments of the present invention include all solvates of the compounds described herein. As used herein, the term "solvate" refers to an aggregate containing one or more molecules of the compound of the present invention and one or more solvent molecules. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the present invention can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvated forms. The compounds of the present invention can be true solvates, while in other cases, the compounds of the present invention can simply retain extraneous water or another solvent, or a mixture of water and some extraneous solvent.

[0053] Embodiments of the compounds of the present invention (e.g., compounds of Structure I or II), or salts, tautomers, or solvates thereof, may contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined with respect to absolute stereochemistry as (R) or (S), or for amino acids as (D) or (L). Embodiments of the present invention are intended to encompass all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (−), (R) and (S), or (D) and (L) isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from appropriate optically pure precursors, or resolution of the racemate (or a salt or derivative of the racemate) using, for example, chiral high-pressure liquid chromatography (HPLC).

[0033] When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0054] "Stereoisomers" refer to compounds composed of the same atoms joined by the same bonds, but having different three-dimensional structures that are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another. "Tautomer" refers to the migration of a proton from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any compound. Various tautomeric forms of a compound can be readily derived by one skilled in the art. The chemical naming protocols and structure diagrams used herein are modifications of the IUPAC nomenclature using the ACD / Name Version 9.07 software program and / or the ChemDraw Ultra Version 11.0 software naming program (CambridgeSoft). Common names familiar to those skilled in the art are also used.

[0055] As mentioned above, one embodiment of the present invention provides a compound useful as a nucleotide probe in various analytical methods. Generally, the present application is directed to a compound comprising at least two fluorophores or fluorophore quenchers covalently attached to a polynucleotide. Without wishing to be bound by theory, it is believed that the spacer between the fluorophores helps maintain sufficient spatial distance between the fluorescent moieties so that intramolecular quenching is reduced or eliminated, resulting in a dye compound with high fluorescence emission.

[0056] Thus, in some embodiments, compounds of the present disclosure have the following structure (I) or a stereoisomer, salt, or tautomer thereof: [ka] (In the formula, M is, independently in each occurrence, either a) the same or different fluorophores, or b) the same or different fluorophore quenchers; L 1a is, independently in each occurrence, a heteroarylene linker; L 2 and L 8 are independently an optional linker,

[0057] L 1b , L 3 , L 5 , L 6 and L 7is independently in each occurrence any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 is independently in each occurrence an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; R 1 and R 2 each independently comprises a polynucleotide, or R 1 and R 2 comprises a polynucleotide, each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 3 is independently in each occurrence H, alkyl, or alkoxy; R 4 is independently in each occurrence OH, SH, O-, S-, ORd, or SRd; R 5 is independently in each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O-, S-, OR d or SR d and

[0058] R c OH, SH, O - , S - , OR d , OL', SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, L' is, independently in each occurrence, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, or a linker comprising a covalent bond to a nucleoside; m, independently in each occurrence, is an integer greater than or equal to zero; n is an integer equal to or greater than 1, q and w are independently 0 or 1 in each occurrence if at least one occurrence of either q or w is 1.

[0059] In some embodiments, M 1 and M 2 are the same or different fluorophores. In other embodiments, M1 and M2 are the same or different fluorophore quenchers. In embodiments, R1 comprises a polynucleotide. 2 comprises a polynucleotide. In various embodiments, the polynucleotide has a sequence that is at least 90% complementary to a target nucleotide sequence.

[0060] In other embodiments, the compositions of the present disclosure comprise a compound of structure (Ia) and a compound of structure (Ib), or stereoisomers, salts, or tautomers thereof. [ka] (In the formula, M 1 are, independently in each occurrence, the same or different fluorophores, M 2 is, independently in each occurrence, the same or different fluorophore-quencher;

[0061] L 1a is, independently in each occurrence, a heteroarylene linker; L 2 and L 8 are independently an optional linker, L 1b , L 3 , L 5 , L6 and L 7 is independently in each occurrence any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 is independently in each occurrence an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; R 1a and R 2b each independently comprises a polynucleotide, or R 1a and R 2b comprises a polynucleotide, each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 1b and R 2a each independently comprises a polynucleotide, or R 1a and R 2b comprises a polynucleotide, each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 3 is independently in each occurrence H, alkyl, or alkoxy;

[0062] R 4 is independently in each occurrence OH, SH, O-, S-, OR d or SR d and R 5 is independently in each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O-, S-, OR d or SR dand R c OH, SH, O - , S - , OR d , OL', SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, L' is, independently in each occurrence, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, or a linker comprising a covalent bond to a nucleoside; m, independently in each occurrence, is an integer greater than or equal to zero; n is an integer equal to or greater than 1, q and w are independently 0 or 1 in each occurrence if at least one occurrence of either q or w is 1.

[0063] In embodiments, R 1a and R 2a each comprise a first and a second polynucleotide, the first polynucleotide comprising 4 to 40 nucleotides (e.g., 6 to 40, 10 to 20, 15 to 30, etc.), the first polynucleotide having a first sequence, the second polynucleotide having a second sequence, and the first sequence having at least 90% complementarity to at least a portion of the second sequence. 1b and R 2b each comprise a third and a fourth polynucleotide, the third polynucleotide comprising 4 to 40 nucleotides (e.g., 6 to 40, 10 to 20, 15 to 30, etc.), the third polynucleotide having a third sequence, and the fourth polynucleotide having a fourth sequence, the third sequence having at least 90% complementarity to at least a portion of the fourth sequence.

[0064] In embodiments, R 1a and R 1beach comprise a first and a second polynucleotide, the first polynucleotide nucleotide sequence comprising 4 to 40 nucleotides (e.g., 6 to 40, 10 to 20, 15 to 30, etc.), the first polynucleotide having a first sequence, the second polynucleotide having a second sequence, and the first sequence having at least 90% complementarity to at least a portion of the second nucleotide sequence. 2a and R 2b each comprise a third and a fourth polynucleotide, the third polynucleotide comprising 4 to 40 nucleotides, the third polynucleotide having a third sequence, and the fourth polynucleotide having a fourth sequence, the third sequence having at least 90% complementarity to at least a portion of the fourth sequence. In embodiments, R 1b , R 2a Or both have a nucleotide sequence that is at least 90% complementary to the target nucleotide sequence. In various embodiments, the second sequence, the third sequence, or both, have at least 90% complementarity to the target nucleotide sequence.

[0065] In other embodiments, the compounds of the present disclosure have the following structure (II) or a stereoisomer, salt, or tautomer thereof: [ka] (II) (In the formula, M 1 are, independently in each occurrence, the same or different fluorophores, M 2 is, independently in each occurrence, the same or different fluorophore-quencher;

[0066] L 1a is, independently in each occurrence, a heteroarylene linker; L 2 and L 8 are independently an optional linker, L 1b , L3 , L 5 , L 6 and L 7 is independently in each occurrence any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 is independently in each occurrence an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 9 is a linker comprising a polynucleotide, R 1a and R 2a each independently comprises a polynucleotide, R 1b and R 2b each independently comprise a polynucleotide or each independently comprise H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 3 is independently in each occurrence H, alkyl, or alkoxy; R 4 is independently in each occurrence OH, SH, O-, S-, OR d or SR d and

[0067] R 5 is independently in each occurrence oxo, thioxo, or absent; R a is O or S, R b is OH, SH, O-, S-, ORd or SRd, R c OH, SH, O - , S - , OR d , OL', SR d, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, L' is, independently in each occurrence, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, or a linker comprising a covalent bond to a nucleoside; m, independently in each occurrence, is an integer greater than or equal to zero; n is an integer equal to or greater than 1, q and w are independently 0 or 1 in each occurrence if at least one occurrence of either q or w is 1.

[0068] In embodiments, R 1a and R 2a each comprise a first and a second polynucleotide, the first polynucleotide comprising 4 to 40 nucleotides, the first polynucleotide having a first sequence, and the second polynucleotide having a second sequence, the first sequence having at least 90% complementarity to at least a portion of the second sequence. 1b and R 2b each comprise a third and a fourth polynucleotide, the third polynucleotide comprising 4 to 40 nucleotides, the third polynucleotide having a third sequence, and the fourth polynucleotide having a fourth sequence, the third sequence having at least 90% complementarity to at least a portion of the fourth sequence. In an embodiment, L 9comprises a fifth polynucleotide having at least 90% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 92% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 95% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 97% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 98% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 99% complementarity to the target nucleotide sequence.

[0069] In further embodiments, the compounds of the present disclosure have the following structure (III) or a stereoisomer, salt, or tautomer thereof: [ka] (III) (In the formula, M 1 are, independently in each occurrence, the same or different fluorophores, M 2 is, independently in each occurrence, the same or different fluorophore-quencher; L 1a is, independently in each occurrence, a heteroarylene linker; L 2 and L 8 are independently an optional linker, L 1b , L 3 , L 5 , L 6 and L 7 is independently in each occurrence any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 is independently in each occurrence an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L9 is a linker comprising a polynucleotide,

[0070] R 1a and R 2a each independently comprise a polynucleotide or each independently comprise H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a )(R b )R c and R 1b and R 2b each independently comprises a polynucleotide, R 3 is independently in each occurrence H, alkyl, or alkoxy; R 4 is independently in each occurrence OH, SH, O-, S-, OR d or SR d and R 5 is independently in each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O-, S-, OR d or SR d and R c OH, SH, O - , S - , OR d , OL', SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, L' is, independently in each occurrence, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, or a linker comprising a covalent bond to a nucleoside; m, independently in each occurrence, is an integer greater than or equal to zero; n is an integer equal to or greater than 1, q and w are independently 0 or 1 in each occurrence if at least one occurrence of either q or w is 1.

[0071] In embodiments, R 1b and R 2b each comprise a first and a second polynucleotide, the first polynucleotide comprising 4 to 40 nucleotides, the first polynucleotide having a first sequence, and the second polynucleotide having a second sequence, the first sequence having at least 90% complementarity to at least a portion of the second sequence. 1a and R 2a each comprise a third and a fourth polynucleotide, the third polynucleotide comprising 4 to 40 nucleotides, the third polynucleotide having a third sequence, and the fourth polynucleotide having a fourth sequence, the third sequence having at least 90% complementarity to at least a portion of the fourth sequence.

[0072] The polynucleotide in any of structures (I), (II), or (III) may terminate in any acceptable group when at the terminal portion of the compound. For example, certain polynucleotides terminate in either a hydroxyl group or a phosphate group. In various other embodiments, the polynucleotide may terminate in -OP(=R a )(R b )R c Ends with R c is OL' and R a and R b is as defined above. In some of these embodiments, L' is a heteroalkylene linker to a solid support, a solid support residue, or a nucleoside. In some embodiments, L' comprises an alkylene oxide or a phosphodiester moiety, or a combination thereof. In certain embodiments, L' has the following structure: [ka] (In the formula, m" and n" are independently integers from 1 to 10; R e is H, an electron pair, or a counterion, L” is R e or a direct bond or linkage to a solid support, solid support residue, or nucleoside (e.g., deoxythymidine)

[0073] In some embodiments, the polynucleotide ends with the following structure: [ka] (wherein dT is deoxythymidine) In embodiments, L9 comprises a fifth polynucleotide having at least 90% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 92% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 92% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 95% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 97% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 98% complementarity to the target nucleotide sequence. In embodiments, the fifth polynucleotide has at least 99% complementarity to the target nucleotide sequence.

[0074] In embodiments, each occurrence of q is 0. In embodiments, at least one occurrence of q is 1. In embodiments, each occurrence of w is 0. In embodiments, at least one occurrence of w is 1. In embodiments, each occurrence of q is 0. In embodiments, L 4 is heteroalkylene, or each occurrence of L is heteroalkylene. In embodiments, heteroalkylene comprises alkylene oxide. In other embodiments, heteroalkylene comprises ethylene oxide. In some embodiments, L has the following structure: [ka] (In the formula, z is an integer from 1 to 100, * indicates a bond to the adjacent phosphorus atom)

[0075] In an embodiment, z is an integer of 3 to 6 or an integer of 22 to 26. In an embodiment, L 4 at least one occurrence of is alkylene, or L 4 is alkylene. In various embodiments, at least one alkylene is ethylene, or each alkylene is ethylene. In further embodiments, R 3 At least one occurrence of is H or R 3 is H. In some embodiments, L 1a is independently in each occurrence an optionally substituted 5- to 7-membered heteroarylene linker. In embodiments, L1a has one of the following structures: [ka]

[0076] In an embodiment, L 3 is an alkylene linker, or L 3 is an alkylene linker. 2 and / or L 8 At least one occurrence of is absent or L 2 and / or L 8 In an embodiment, each occurrence of L 5 and / or L 6 at least one occurrence of is alkylene, or L 5 and / or L 6 Each occurrence of L is alkylene. 1b independently in each occurrence includes an amide functionality or a triazolyl functionality. In embodiments, R 5 independently in each occurrence, OH, O - OR d In an embodiment, R 4 is oxo in each occurrence.

[0077] In an embodiment, L 7 at least one occurrence of is an optionally substituted heteroalkylene linker, or L 7 Each occurrence of is independently an optionally substituted heteroalkylene linker. 7 In some embodiments, L comprises an amide functional group or a triazolyl functional group. 7 Each occurrence of has one of the following structures: [ka] In an embodiment, n is an integer from 1 to 100, or n is an integer from 1 to 10, or 2 to 10. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In an embodiment, m is an integer from 3 to 6, or m is 3.

[0078] In embodiments, the fluorophore, independently at each occurrence, is a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, 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. In embodiments, the fluorophore is, independently at each occurrence, pyrene, perylene, perylene monoimide, 5-FAM or 6-FAM, or a derivative thereof. In further embodiments, the fluorophore is independently selected at each occurrence from Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0079] In certain embodiments, the fluorophore has, independently at each occurrence, one of the following structures: [ka]

[0080] In embodiments, the fluorophore has, independently at each occurrence, one of the following structures: [ka] [ka] [ka] [ka] [ka]

[0081] In further embodiments, the fluorophore quencher is independently selected at each occurrence from Table 2. [Table 2-1] [Table 2-2] [Table 2-3]

[0082] In some embodiments, the fluorophore quencher is, independently at each occurrence, BHQ-1 or BHQ-2. In other embodiments, the fluorophore quencher is independently at each occurrence: [ka] [ka] [ka] IRDye QC-1; Iowa Black FQ; Iowa Black RQ; OSY35; OSY7; OSY21; Cy5Q; Cy7Q; QXL 490; QXL 52 0; QXL 570; QXL 610; QXL 670; QXL 680; ATTO 5 40Q; ATTO 580Q or ATTO 612Q.

[0083] As will be appreciated, the compounds described herein (including the detectable probe and quencher constructs) may be formed using any suitable method, such as those described in U.S. Patent Publication Nos. 2017 / 0292957, 2016 / 0208100, 2016 / 0341736, 2018 / 0065998, 2018 / 0079909, and 2019 / 0016898, which are incorporated by reference in their entireties for such teachings. Schematic versions of an illustrative detectable probe and an illustrative quencher molecule are shown in Figure 1A. As shown, the polynucleotides on the left are complementary and therefore hybridize to bring the fluorophore and quencher into close proximity. While a perfectly complementary polynucleotide is shown in Figure 1A, some mismatched nucleotide pairs may be found in the polynucleotide, as shown in Figure 1B. While the degree of complementarity is less than 100%, this is still sufficient to bring the fluorophore and quencher into close proximity.

[0084] Additionally, polynucleotides may be attached to either side of the fluorophore and / or quencher moieties. A schematic representation of a pair of such constructs is shown in Figure 1C. The sequences of the polynucleotides on either side of the fluorophore and quencher moieties are sufficiently complementary to hybridize, thereby bringing the fluorophore and quencher into close proximity. In a further embodiment, the detectable probe comprises a fluorophore and a quencher moiety, as schematically illustrated in Figure ID. The portion of the polynucleotide complementary to the target sequence is found at the nucleotide of the loop structure on the left. While the polynucleotide is shown attached to both the fluorophore and quencher moieties, in other embodiments, the polynucleotide is attached to one side of the fluorophore and quencher moieties (similar to the construct in Figure IA).

[0085] Embodiments of the present disclosure also include a detectable probe for identifying the presence of a target nucleotide sequence, the detectable probe comprising: a first polynucleotide having a first end, the first sequence comprising a target-complementary sequence having at least 90% complementarity to the target nucleotide sequence; a first polymer having a first end and a second end, the first end covalently attached to the first end of the first polynucleotide, the first polymer comprising two fluorophores; and a second polynucleotide having a first end covalently attached to the second end of the first polymer. In some embodiments, the detectable probe further comprises a second polymer having a first end and a second end, the first end being covalently attached to the second end of the first polynucleotide, and a third polynucleotide having a first end covalently attached to the second end of the second polymer.

[0086] In various embodiments, the second polynucleotide has a second sequence that includes a first regulatory complementary sequence, and the third polynucleotide has a third sequence that includes a second regulatory complementary sequence that has at least 75% complementarity to the first regulatory complementary sequence. In various embodiments, such compositions or kits further comprise a quencher molecule comprising a third polynucleotide having a first end, a second polymer having a first end and a second end, the first end being covalently attached to the first end of the third polynucleotide, the second polymer comprising two fluorophores, and a fourth polynucleotide having a first end covalently attached to the second end of the second polymer. In various embodiments, the first sequence further comprises a first regulatory complementary sequence, and the third polynucleotide has a third sequence comprising a second regulatory complementary sequence capable of hybridizing to the first regulatory complementary sequence. In some such embodiments, the first and second regulatory sequences are complementary, as depicted in Figure 1A. In other embodiments, there are one or more mismatched nucleotides between the first and second regulatory sequences, as depicted in Figure 1B. In a further embodiment, the second polynucleotide has a second sequence that includes a third regulatory complementary sequence, and the fourth polynucleotide has a fourth sequence that includes a fourth regulatory complementary sequence that is capable of hybridizing to the third regulatory complementary sequence.

[0087] In any of the above embodiments, the target complementary sequence is capable of hybridizing to the target nucleotide sequence at a first intensity, and the first regulatory complementary sequence is capable of hybridizing to the second regulatory complementary sequence at a second intensity that is less than the first intensity. Various embodiments of the present disclosure provide a detectable probe for identifying the presence of a target nucleotide sequence, comprising: a) a first polynucleotide covalently attached to a first polymer comprising two or more fluorophores; b) a second segment comprising a second nucleotide sequence covalently attached to a second polymer comprising two or more fluorophore quenchers; Including, the first nucleotide sequence comprises: i) a target-complementary sequence having at least 90% complementarity to a target nucleic acid sequence, capable of forming a double-stranded hybrid with the target sequence under assay conditions having a first strength; and ii) a probe-complementary sequence having at least 90% complementarity to at least a portion of a second nucleotide sequence, capable of forming a double-stranded hybrid with at least a portion of the second nucleotide sequence under assay conditions having a second strength, the second strength being less than the first strength;

[0088] The first polymer further comprises a detectable probe that, in the absence of the second polymer, has a peak fluorescence emission when excited with ultraviolet light of a predetermined wavelength that is at least 85% of the sum of the peak fluorescence emissions of the individual fluorophores present in the first polymer when excited with ultraviolet light of the same wavelength. In some such embodiments, the first nucleotide sequence is covalently linked to the second nucleotide sequence. Also described herein are compositions comprising the described detectable probes, as well as kits comprising such compositions. In some such embodiments, the disclosed kits further comprise instructions for using the compounds, compositions, or detectable probes to identify target nucleotide sequences.

[0089] Further described are methods for identifying the presence of a target nucleotide sequence, comprising contacting a sample under assay conditions with a compound, composition, or detectable probe described herein to produce a mixture, and imaging the mixture under detection conditions. In some embodiments, compounds of the present disclosure have the structures shown in Table 3. It should be understood that the structures shown in the tables below are meant to be representative examples of embodiments of the present invention. In certain embodiments, the compounds of Table 3 are fluorescent probe compounds, and the M moieties, independently in each occurrence, are the same or different fluorophores. In more specific embodiments, the compound of structure III-1, III-2, III-3, III-IV, or III-V in Table 3 is a fluorescent probe molecule, and the M moiety is a fluorophore. In more specific embodiments, the fluorophore is selected from Table 1.

[0090] In other embodiments, the compounds of Table 3 are fluorescence quenching compounds, and the M moieties, independently in each occurrence, are the same or different fluorophore quenchers. In even more specific embodiments, the compound of structure III-6, III-7, III-8, III-9, or III-10 in Table 3 is a fluorescent quencher molecule, and the M moiety is a fluorophore quencher. In more specific embodiments, the fluorophore quencher is selected from Table 2. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] As used in Tables 3, 4 and 5 and throughout this application, R 2 , R 3 , m, n, and L' have the definitions provided for the compound of structure (I) unless otherwise indicated, F refers to a fluorescein moiety having the structure shown in Table 1, and BHQ1 and DAB refer to quencher moieties having the structure shown in Table 2.

[0091] "dT" refers to the following structure: [ka] [Example]

[0092] General method Mass spectral analysis was performed on a Waters / Micromass Quattro micro MS / MS system (MS-only mode) using MassLynx 4.1 acquisition software. The mobile phase used for LC / MS of the dyes was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8. The phosphoramidite and precursor molecules were also analyzed using a Waters Acquity UHPLC system equipped with a 2.1 mm x 50 mm Acquity BEH-C18 column maintained at 45 °C using an acetonitrile / water mobile phase gradient. The molecular masses of the monomer intermediates were obtained using a Waters / Micromass Quattro micro MS / MS system (MS-only mode) using tropylium cation infusion-enhanced ionization. Excitation and emission profile experiments were recorded on a Cary Eclipse spectrophotometer.

[0093] All reactions were performed in oven-dried glassware under a nitrogen atmosphere unless otherwise noted. Commercial DNA synthesis reagents were purchased from Glen Research (Sterling, VA). Anhydrous pyridine, toluene, dichloromethane, diisopropylethylamine, triethylamine, acetic acid, pyridine, and THF were purchased from Aldrich. All other chemicals were purchased from Aldrich or TCI and used as is without further purification.

[0094] Example 1 Synthesis of dyes with ethylene glycol spacers Compounds with ethylene oxide linkers were prepared as follows. Oligofluoroside constructs (e.g., compounds of structure (I), (II), or (III)) were synthesized on a 1 μmol-scale Applied Biosystems 394 DNA / RNA synthesizer and contained either a 3'-phosphate group or a 3'-S2-(CH2)6-OH group, or other groups described herein. Synthesis was performed directly on CPG beads or polystyrene solid supports using standard phosphoramidite chemistry. Oligofluorosides were synthesized in the 3' to 5' direction using standard solid-phase DNA methods, coupling with standard β-cyanoethyl phosphoramidite chemistry. The phosphoramidites of fluorosides and nucleosides, as well as spacers (e.g., hexaethyloxy-glycol phosphoramidite, triethyloxy-glycol phosphoramidite, polyethylene glycol phosphoramidite), and linkers (e.g., 5'-amino-modifier phosphoramidite and thiol-modifier S2 phosphoramidite) were dissolved in acetonitrile to make a 0.1 M solution and sequentially added using the following synthesis cycle: 1) removal of the 5'-dimethoxytrityl protecting group with dichloroacetic acid in dichloromethane, 2) coupling of the next phosphoramidite with an activating reagent in acetonitrile, 3) oxidation of P(III) with iodine / pyridine / water to form stable P(v), and 4) coupling of any unreacted 5'-hydroxyl groups with acetic anhydride / 1-methylimidazole / acetonitrile. The synthesis cycle was repeated until the full-length oligofluoroside construct was assembled. At the end of chain assembly, the monomethoxytrityl (MMT) or dimethoxytrityl (DMT) groups were removed with dichloroacetic acid in dichloromethane.

[0095] Compounds were provided at a 0.2 μmol scale on controlled-pore glass (GCP) supports in labeled Eppendorf tubes. 400 μL of 20-30% NH4OH was added and mixed gently. The open tubes were placed at 55°C for approximately 5 minutes, or until excess gas was released, then sealed and incubated for 2 hours (+ / - 15 minutes). The tubes were removed from the heat block, allowed to reach room temperature, and then centrifuged at 13,400 RPM for 30 seconds to solidify the supernatant and solids. The supernatant was carefully removed and placed in a labeled tube, and 150 μL of acetonitrile was then added to wash the support. After adding the wash solution to the tubes, the tubes were placed in a CentriVap instrument at 40°C until dry. The product was characterized by ESI-MS, UV absorbance and fluorescence spectroscopy.

[0096] Example 2 Spectral testing of compounds The dried compound was reconstituted with 150 μL of 0.1 M Na2CO3 buffer to make a stock solution of approximately 1 mM. The concentrated stock solution was diluted 50x with 0.1x PBS and analyzed by a NanoDrop UV spectrometer to obtain an absorbance reading. The absorbance reading was used in conjunction with the extinction coefficient (75,000 M-1cm-1 for each FAM unit) and Beer's Law to determine the actual concentration of the stock solution. From the calculated stock concentrations, approximately 4 mL of 5 μM solutions were made in 0.1 M Na2CO3 (pH 9) and analyzed in 1 x 1 cm quartz cuvettes with a Cary 60 UV spectrometer using the spectral range of 300 nm to 700 nm to measure overall absorbance for the group. From these 5 μM solutions, a second dilution was made at 50 nM or 25 nM (also in 0.1 M Na2CO3, pH 9) for spectroscopic analysis with a Cary Eclipse Fluorimetry instrument. Excitation was set at 494 nm, and emission spectra were collected from 499 to 700 nm.

[0097] Example 3 Melting curve analysis of probes in the presence of quencher constructs with complementary oligomer sequences. Probe compounds P-1 and P-3, containing 20-mer oligonucleotide sequences, were synthesized according to the method described in Example 1 herein. Probes P-1 and P-3 each contain one or two fluorescein moieties. P-1 and P-3 were prepared in solution at a concentration of 2 μM and incubated in the presence or absence of quencher construct Q-4, containing a complementary 20-mer oligonucleotide sequence and a single BHQ-1 moiety. In tests where the complementary probe and quencher compounds were mixed, the complementary oligonucleotides aligned to form a hybrid complex. Samples were incubated at ambient temperature for 10 minutes and then analyzed using a CFX-96 thermal cycler to generate melting curves. Figures 2 and 3 show the melting curves of probes P-1 and P-3 using a quencher concentration of 6.4 μM (Figure 2) or 9.1 μM (Figure 3) of Q-4. A reduction in fluorescence was observed in the probe / quencher mixture compared to the sample containing only the probe; see initial fluorescence at 27 °C. Compound P-3, which has two fluorescein moieties, demonstrated an approximately 1.6-fold increase in fluorescence over P-1, which contains a single fluorescein moiety. Denaturation of the hybrid complex can be observed to begin at approximately 50 °C, with fluorescence increasing as the temperature increases. The increase in fluorescence is a result of denaturation, since the quencher moiety increases the average distance away from the fluorophore, demonstrating minimal efficiency. The results also demonstrate a strong temperature dependence of the observed fluorescence of the probe in the absence of a quencher, as a result of dynamic quenching of fluorescence due to temperature-related changes in the mobility of groups within the compound.

[0098] Example 4 Melting curve analysis of the probe in the presence of a quencher construct with the same non-complementary oligomer sequence. In a negative control experiment, the probe and a quencher compound with a non-complementary oligonucleotide sequence were incubated together to demonstrate the lack of hybridization and, consequently, the absence of significant quenching. Probe compound P-2, containing a 20-mer oligonucleotide sequence, was synthesized according to the method described in Example 1 herein and contains a single fluorescein moiety. P-2 was prepared in solution at a concentration of 2 μM and incubated in the presence or absence of quencher construct Q-4 (6 μM), which contains the same non-complementary 20-mer oligonucleotide sequence. Therefore, in mixed sample analysis, no hybrid complex formation should occur, and no increase in fluorescence as a result of hybrid complex denaturation should occur. Samples were incubated at ambient temperature for 10 minutes and then analyzed using a CFX-96 thermal cycler to generate melting curves ranging from an initial temperature of 27°C to a final temperature of 80°C. The melting curve analysis depicted in Figure 4 shows the absence of a fluorescence increase in the P-2 / Q-4 mixture, demonstrating the lack of quantifiable hybridization as a result of the identical non-complementary oligonucleotide sequences. A decrease in fluorescence was observed in the P-2 / Q-4 mixture compared to the analysis of P-2 alone, which is attributed to intermolecular quenching.

[0099] Example 5 Determining the relative quenching efficiency of probes in the presence of quencher constructs To eliminate the observed changes in fluorescence with increasing temperature and facilitate interpretation of the curves, analysis of the fluorescence data generated in Examples 3 and 4 was performed to determine the relative quenching efficiency across the melting curve analysis experiments. Figure 5 shows the relative quenching efficiencies of probe compounds P-1, P-2, and P-3 as a function of temperature in the presence of the quenching probe Q-4. As expected, the data for P-2 in the presence of the non-complementary quencher are essentially constant at 20%, since there is no denaturation upon heating; intermolecular quenching is observed only in the non-hybrid complex. Interestingly, the relative quenching efficiencies of P-1 and P-3 are similar despite the difference in the number of fluorescein moieties (one and two, respectively) and the varying concentrations of Q-4 (6.4 and 9.1 μM). It is noteworthy that Q-4, which contains a single BHQ-1 moiety, had a quenching efficiency similar to both P-1 and P-3, despite the difference in the number of fluorescein moieties.

[0100] Example 6 Determining the fluorescence of probes with increasing numbers of fluorescein moieties Probe compounds P-1, P-4, P-5, P-6, and P-7, containing 20-mer oligonucleotide sequences and containing one, two, three, four, and five fluorescein (F) moieties, respectively, were synthesized according to the method described in Example 1 herein. Maximum fluorescence was determined according to the method described in Example 1 herein. The results are summarized in Table 6. [Table 6] The results demonstrate that the increase in fluorescence was not directly proportional to the number of fluorescein moieties; see, for example, Table 6, where the fluorescence of P-4, which has two F moieties, did not exhibit twice the fluorescence compared to P-1, which has one F moiety. The non-proportional increase in fluorescence is due to intramolecular quenching phenomena caused by the increase in polymer length as a result of the incorporation of additional fluorophores.

[0101] Example 7 Quenching of probes with various fluorescein moieties using a quencher probe containing four BHQ-1 moieties, including melting curve analysis Probe compounds P-1, P-4, P-5, P-6, and P-7, containing 20-mer oligonucleotide sequences and containing one, two, three, four, and five fluorescein moieties, respectively, were synthesized as described above. Solutions of the test compounds were prepared at a concentration of 0.5 μM according to the method described in Example 2 herein. Prior to analysis, samples were heated to 80°C for 2 minutes and then cooled to ambient temperature for 5 minutes. These test solutions were used for melting curve analysis of the individual probe compounds (see below). The maximum fluorescence of each probe, P-1, P-4, P-5, P-6, and P-7, was determined from the initial measurements of the melting curve analysis.

[0102] Separate 0.5 μM solutions of test compounds were incubated with 1.0 μM of the quenching probe Q-6, which contains four BHQ-1 moieties, for 10 minutes at ambient temperature. Prior to analysis, samples were heated to 80° C. for 2 minutes and then cooled to ambient temperature for 5 minutes before performing melting curve analysis. Quenched fluorescence measurements were determined from the initial melting curve analysis readings. Figure 6 shows bar graphs of the fluorescence observed for compounds P-1, P-4, P-5, P-6, and P-7 in the absence (left bar) and presence (right bar) of quencher Q-6. As expected, the quenching efficiency observed for Q-6 (BHQ-1 × 4) decreased with increasing number of fluorescein (F) moieties in the probe, with the highest quenching observed for the mono-F compound P-1 (82.4%) and the lowest for the penta-F compound P-7 (58.0%). It is noteworthy from this data that the increase in fluorescence was minimal when the number of fluorescein moieties was increased from 4 to 5, suggesting that intramolecular quenching in this series of compounds may limit overall brightness once the compound reaches a certain length.

[0103] Melting curve analysis of the samples prepared above was performed using a CFX-96 thermal cycler ramping from an initial temperature of 0°C to a final temperature of 122°C. Figure 7 shows the melting curve analysis of all test samples. Consistent with the results of Example 3, test compounds lacking the quencher probe exhibited a strong temperature dependence of the observed fluorescence. Furthermore, the increase in fluorescence observed with the probe / quencher combination indicates that the hybrid complex is denaturing. The onset of the method was consistent across all test samples, occurring at approximately 50°C, suggesting some robustness in the nature of the hybrid complex. Upon denaturation, the fluorescence properties of the combined test solution closely mimicked those of the probe-only solution.

[0104] Example 8 Demonstration of probe and quencher hybridization upon cooling using a thermal cycler Solutions of probe compounds P-1 and P-6, and probe P-8, were prepared and analyzed using a thermal cycler in the cooling mode (warm-to-cool transition) in the presence of complementary quenchers Q-6 (4 × BHQ-1 moieties) and Q-7 (4 × D ab moieties), respectively. Quencher-free solutions of each probe compound were also prepared and analyzed in the same manner. P-1 contains one fluorescein moiety, while P-6 and P-8 each contain four. Test solutions were heated to 80°C in a CFX-96 thermal cycler, held for 2 minutes, and then cooled to 25°C (-5°C / cycle, 4 second hold).

[0105] A plot of probe fluorescence as a function of temperature is shown in Figure 8. As expected in the cooling mode, the probe-only solution demonstrated an increase in fluorescence as the temperature decreased, while the opposite trend was observed in the melting curve analysis. Similarly, test samples of the probe and quencher, respectively, demonstrated a decrease in fluorescence as the complementary oligonucleotides began to anneal and form hybrid complexes (schematically shown in Figures 1A and 1C). Without wishing to be bound by theory, Applicants believe that the large reduction in fluorescence relative to the initial state (80°C) of the P-8 / Q-7 hybrid compared to the P-6 / Q-6 hybrid results from improved quenching ability as a result of the quenching moiety coming into close proximity to the fluorophore, or superior hybridization efficiency, or a combination of both.

[0106] Example 9 Comparison of probe-pair quenching efficiencies for probe pairs Probe compounds P-1 and P-6, and probe P-8, were synthesized as described above. Solutions of test compounds were prepared at a concentration of 1.0 μM according to the method described in Example 2 herein. Prior to analysis, samples were heated to 80°C for 2 minutes and then cooled to ambient temperature for 5 minutes. These test solutions were then used for melting curve analysis of the individual probe compounds, see below. The maximum fluorescence of individual probes P-1, P-6, and P-8 was determined from the initial measurements of the melting curve analysis. P-1 contains one fluorescein moiety, while P-6 and P-8 each contain four. Separate 1.0 μM solutions of test compounds were incubated with 1.9 μM quenching probe Q-6 for probes P-1 and P-6, and quencher Q-7 for probe P-8 at ambient temperature for 10 minutes. Prior to analysis, samples were heated to 80° C. for 2 minutes and then cooled to ambient temperature for 5 minutes before melting curve analysis was performed. Quenched fluorescence measurements were also determined from the initial melting curve measurements.

[0107] Figure 9 shows a bar graph of the fluorescence observed for compounds P-1, P-6, and P-8 in the absence (left bar) and presence (right bar) of quenchers Q6 or Q7, as indicated. Surprisingly, the P-8 / Q-7 pair demonstrated an 81% reduction in fluorescence, a value comparable to the P-1 / Q-6 pair (82%), which has only a single fluorescein moiety in P-1 versus four quenching moieties in Q-6. In contrast, the P-8 probe contains four fluorescein moieties, equal to the number of quenching moieties in Q-7. Additional test solutions of P-8 / Q-7 were prepared to investigate quenching efficiency at higher concentrations. Test solutions of P-8 were prepared at 4.3 and 8.6 μM and analyzed in a manner similar to determining the fluorescence of the individual probe solutions. Similarly, separate solutions of the test compounds were incubated with the quenching probe Q-7, this time at a concentration of 10.6 μM, for 10 minutes at ambient temperature. These results are also depicted in FIG. 9, and a summary of the data can be found in Table 7.

[0108] It is noted from these data that a further increase in quenching was observed at higher probe concentrations (4.3 and 8.6 μM), reaching nearly 90% even at low quencher / probe ratios, see last entry in Table 7. [Table 7] By design, probe and quencher pairs with polynucleotide sequences flanking the fluorophore and quencher are intended to bring one or more fluorophores of the quencher probe into close proximity with the quencher moiety of a complementary quencher polymer through hybridization occurring on either side of the fluorescent and quencher moieties (see Figure 1C). Without being bound by theory, Applicant believes that the pair variant (see Figure 1A) demonstrates reduced quenching efficiency as a result of the increased distance between the fluorophore moiety and the quencher moiety caused by electrostatic repulsion between the negatively charged phosphate-containing polymer backbones. The probe overcomes this deficiency through tandem hybridization on either side, thereby reducing the distance between the fluorophore and quencher and increasing efficiency. Melting curve analysis of the 1.0 μM samples prepared above was performed using a CFX-96 thermal cycler ramping from an initial temperature of 22°C to a final temperature of 80°C. Figure 10 shows the melting curve analysis of all test samples. Consistent with the other melting curve results, test compounds lacking the quencher probe showed a strong temperature dependence in the observed fluorescence, which decreased significantly with increasing temperature. Additionally, the increase in fluorescence observed with the probe / quencher combination indicates that the hybrid complex is denaturing.

[0109] Example 10 Quenching of probes with various fluorescein moieties using a mono-dentate quenching probe containing a single BHQ-1 moiety Test solutions of probe compounds P-1, P-4, P-5, P-6, and P-7 were prepared as described above in Examples 2 and 7 with a nominal concentration of 0.5 μM. Prior to analysis, samples were heated to 80° C. for 2 minutes and then cooled to ambient temperature for 5 minutes. The maximum fluorescence of individual probes P-1, P-4, P-5, P-6, and P-7 was determined at 22° C. in a CFX-96 thermal cycler. Separate 0.5 μM solutions of test compounds were incubated with 1.3 μM (2.6 equivalents) of the quenching probe Q-4, which contains one BHQ-1 moiety, for 10 minutes at ambient temperature. Prior to analysis, samples were heated to 80°C for 2 minutes and then cooled to ambient temperature for 5 minutes. Quenched fluorescence measurements were determined at 22°C in a CFX-96 thermal cycler.

[0110] Figure 11 shows bar graphs of the fluorescence observed for compounds P-1, P-4, P-5, P-6, and P-7 in the absence (left bar) and presence (right bar) of the quencher Q-4. As expected, the quenching efficiency observed for the probe / Q-4 pair decreased with increasing number of fluorescein (F) moieties in the probe. However, optimal quenching was observed for the di-F compound P-4 (78%), with a similar efficiency for the mono-F compound P-1 (75%). The penta-F compound P-7 exhibited the lowest quenching efficiency (42%). To investigate the effect of lower concentrations of quencher probe Q-4, two additional experiments were performed with solutions of P-1 containing 0.85 μM and 0.45 μM quencher probe Q-4 (1.7 and 0.9 equivalents, respectively). Figure 12 shows the results of this analysis, demonstrating that with less than 1 equivalent of quencher probe, even the less favorable mono-, fluorescence quenching can be approximately 50%.

[0111] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this application and / or listed in the Application Data Sheet, including U.S. Patent Application No. 62 / 908,509, filed September 30, 2019, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to utilize concepts from various patents, applications, and publications to provide even further embodiments. These and other changes can be made to the embodiments in light of the description detailed above. Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. A compound having an ethylene oxide linker having the following structure (I), or its stereoisomers, salts, or tautomers. 【Chemistry 1-1】 (I) (In the formula, In each occurrence, M is independently either a) the same or different fluorophores, or b) the same or different fluorophore enchantments. L 1a Each occurrence is independently a substituted, possibly substituted, 5-7 membered ring heteroarylene linker. L 2 and L 8 It is an independent, arbitrary linker, L 1b , L 3 , L 5 , L 6 and L 7 Each occurrence is independently any alkylene having 1 to 12 carbon atoms, alkenylene having 2 to 12 carbon atoms, alkylylene having 2 to 12 carbon atoms, heteroalkylene having 1 to 12 carbon atoms, heteroalkenylene having 2 to 12 carbon atoms, or heteroalkylylene linker having 2 to 12 carbon atoms. L 4 is, independently at each occurrence, an alkylene having 1 to 12 carbon atoms or a heteroalkylene linker having 1 to 12 carbon atoms, At least one occurrence of L4 has the following structure: [Chemistry 1-2] (In the formula, z is an integer between 1 and 100. (* indicates a bond to an adjacent phosphorus atom) R 1 and R 2 Each independently contains a polynucleotide, or R 1 Or R 2 One of them contains a polynucleotide, and the other of R1 or R2 is independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, or -OP(=R a ) (Caution b ) R c And, R 3 In each occurrence, it is independently H, alkyl, or alkoxy. R 4 In each occurrence, OH, SH, O-, S-, OR d or SR d And, R 5 In each occurrence, independently, it is either oxo, thioxo, or absent. R a is either O or S, R b OH, SH, O-, S-, OR d or SR d And, R c OH, SH, O - S - , OR d ,OL',SR d Alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, R d It is a counterion, L' is, independently in each occurrence, a linker containing a covalent bond to a solid support, a linker containing a covalent bond to a solid support residue, or a linker containing a covalent bond to a nucleoside. m is an integer greater than or equal to zero and less than or equal to 6, independently in each occurrence. n is an integer between 2 and 10. (q and w are independently 0 or 1 in each occurrence, and at least one occurrence of w is 1.)

2. The compound or composition according to claim 1, wherein z is an integer from 3 to 6 or an integer from 22 to 26.

3. The compound or composition according to claim 1 or 2, wherein L 1a has one of the following structures. 【Transformation 6】

4. The compound or composition according to any one of claims 1 to 3, wherein at least one appearance of L3 is an alkylene linker, or each appearance of L3 is an alkylene linker.

5. The compound or composition according to any one of claims 1 to 4, wherein the appearance of at least one of L2 and / or L8 is absent, or each appearance of L2 and / or L8 is absent.

6. The compound or composition according to any one of claims 1 to 5, wherein L 1b independently comprises an amide functional group or a triazolyl functional group in each occurrence.

7. The compound or composition according to any one of claims 1 to 6, wherein at least one of L5 and / or L6 is alkylene, or each of L5 and / or L6 is alkylene.

8. The compound or composition according to any one of claims 1 to 6, wherein at least one occurrence of L5 is absent, or each occurrence of L5 is absent.

9. The compound or composition according to any one of claims 1 to 8, wherein at least one occurrence of R3 is H, or each occurrence of R3 is H.

10. The compound or composition according to any one of claims 1 to 9, wherein R 4 is oxo in each occurrence.

11. The compound or composition according to any one of claims 1 to 10, wherein at least one occurrence of L7 is an optionally substituted heteroalkylene linker, or each occurrence of L7 is independently an optionally substituted heteroalkylene linker.

12. The compound or composition according to any one of claims 1 to 11, wherein L7 comprises an amide or triazolyl functional group.

13. The compound or composition according to any one of claims 1 to 12, wherein each appearance of L 7 has one of the following structures. 【Chemistry 2】

14. The compound or composition according to any one of claims 1 to 13, wherein m is an integer from 3 to 6.

15. The compound or composition according to any one of claims 1 to 14, wherein the fluorophore is independently dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthrasyl, squaline, squallium, 9,10-ethynylanthracene, or ter-naphthyl moiety in each appearance.

16. The compound or composition according to any one of claims 1 to 14, wherein the fluorophore is independently pyrene, perylene, perylene monoimide, 5-FAM or 6-FAM, or a derivative thereof, in each appearance.

17. The compound or composition according to any one of claims 1 to 14, wherein the fluorophore independently has one of the following structures in each appearance. 【Transformation 3】

18. The compound or composition according to any one of claims 1 to 14, wherein the fluorophore independently has one of the following structures in each appearance. 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 【Chemistry 4-4】 [Chemistry 4-5]

19. The fluorophore enchanter independently contains BHQ-1, BHQ-2, DAB, in each appearance. 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 The compound or composition according to any one of claims 1 to 14, wherein the compound is IRDye QC-1; Iowa Black FQ; Iowa Black RQ; OSY35; OSY7; OSY21; Cy5Q; Cy7Q; QXL 490; QXL 520; QXL 570; QXL 610; QXL 670; QXL 680; ATTO 540Q; ATTO 580Q or ATTO 612Q.

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