Scaffolded chromophore for nucleic acid detection, method and use thereof

Abstract

A scaffolded chromophore for nucleic acid detection, as well as systems, methods, and uses thereof, are provided. Specific embodiments relate to nucleic acid probes comprising a nucleic acid complementary to a target sequence. The probe further comprises a dye structure ligated to a first end of the nucleic acid, the dye structure comprising a non-conjugated polymer backbone, one or more donor fluorophores ligated to the polymer backbone, and one or more acceptor fluorophores ligated to the polymer backbone, in an energy transfer relationship with the donor fluorophores. Such a probe may further comprise a quencher ligated to a second end of the nucleic acid, in an energy transfer relationship with one or more acceptor fluorophores. Further embodiments include a second nucleic acid probe comprising a second nucleic acid complementary to a different target sequence.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 539,067, filed on 18 September 2023. The disclosure of that application is incorporated herein by reference.

[0002] Introduction Nucleic acid detection has many applications in science and medicine, such as diagnostics and / or environmental monitoring. However, detection relies on fluorescently labeled probes, which can be subject to certain limitations, including lack of sensitivity, faintness, or inability to multiplex. Due to these limitations, probes that can be tuned for increased fluorescence emission and / or multiplexing are needed. [Overview of the project]

[0003] A scaffolded chromophore for nucleic acid detection, as well as systems, methods, and uses thereof, are provided. Specific embodiments relate to nucleic acid probes comprising a nucleic acid complementary to a target sequence. The probe further comprises a dye structure ligated to a first end of the nucleic acid, the dye structure comprising a non-conjugated polymer backbone, one or more donor fluorophores ligated to the polymer backbone, and one or more acceptor fluorophores ligated to the polymer backbone, in an energy transfer relationship with the donor fluorophores. Such a probe may further comprise a quencher ligated to a second end of the nucleic acid, in an energy transfer relationship with one or more acceptor fluorophores. Further embodiments include a second nucleic acid probe comprising a second nucleic acid complementary to a different target sequence.

[0004] The probes of a particular embodiment may include DNA, RNA, and / or locked nucleic acids. Individual bases may include canonical bases, modified bases, base analogs, secondary groove binders, water-solubilizing groups, and / or additional quenchers. The non-conjugated polymer backbone may consist of one or more of the following: peptides, carbohydrates, hydrocarbons, polyethylene glycol polymers, lipids, peptoids, and polynucleotides.

[0005] Further embodiments include methods, systems, kits, and hybridization products comprising the nucleic acid probe described above. Such methods can be used to detect a target nucleic acid and include preparing a reaction mixture comprising a sample and a nucleic acid probe, and monitoring the reaction mixture for a signal released from a dye structure to assay the sample for the presence of the target nucleic acid. Such methods may include carrying out a nucleic acid amplification reaction such as PCR and / or isothermal amplification. Kits may include the probe described above, as well as one or more reagents for carrying out reactions such as nucleic acid amplification reactions. Further embodiments relate to products comprising the probe described above hybridized to a target nucleic acid.

[0006] The system includes a sample chamber for holding the sample, an illumination source directed to strike the sample, and a detector such that the light emitted from the sample strikes the detector. Additional systems may include heating elements such as a heat block and / or water bath. Additional systems may include a user interface that allows selection of temperature, illumination wavelength, detection wavelength, and temperature cycle program.

[0007] This invention can be best understood from the following detailed description in conjunction with the accompanying drawings. The drawings include the following figures. [Brief explanation of the drawing]

[0008] [Figure 1]The present invention provides schematic nucleic acid probes having scaffolded chromophore dye structures and quenchers according to various embodiments of the present invention. [Figure 2] The present invention provides non-limiting examples of nucleic acid probes having scaffolded chromophore pigment structures and quenchers according to various embodiments of the present invention.

[0009] definition As used herein, the terms “chemoselective functional group” and “chemoselective tag” are used interchangeably and, in some cases, refer to a functional group that, after the activation of one of the functional groups of any choice, can selectively react with another compatible functional group to form a covalent bond. The chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their active esters, as well as groups that can react with each other via click chemistry, such as azides and alkynes (e.g., cyclooctin), tetrazines, transcyclooctene, dienes and dienophiles, as well as azides, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, as well as hydroxyls, hydrazides, hydrazinos, aldehydes, ketones, azides, alkynes, phosphines, epoxides, and the like.

[0010] As used herein, the term “sample” refers to one or more analytes of interest, for example, a material or mixture of materials, possibly in liquid form, containing nucleic acids. In some embodiments, the term, as used in its broadest sense, refers to any plant, animal or bacterial material containing cells or producing cellular metabolites, such as tissues or bodily fluids isolated from an individual (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or in vitro cell culture components, as well as samples from the environment. The term “sample” may also refer to a “biological sample.” As used herein, the term “biological sample” refers to an entire organism or a subset of its tissues, cells or components (including, but not limited to, bodily fluids such as blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid and semen). The term “biological sample” also refers to, but is not limited to, homogenates, lysates, or extracts, or fractions or parts thereof, prepared from a whole organism or a subset of its tissues, cells, or components, including plasma, serum, cerebrospinal fluid, lymph, skin, respiratory tract, intestines, and external sections of the urogenital tract, tears, saliva, milk, blood cells, tumors, and organs. In certain embodiments, the sample is taken from an animal or plant. A biological sample may include cells. The term “cell” is used in its conventional sense and refers to the basic structural unit of both eukaryotic and prokaryotic organisms that has at least a nucleus and a cell membrane. In certain embodiments, cells include prokaryotic cells, such as those derived from bacteria. In other embodiments, cells include eukaryotic cells, such as those obtained from a biological sample derived from an animal, plant, or fungus. The term “sample” may further refer to “environmental sample.” As used herein, the term “environmental sample” refers to a swab taken from a non-biological location (a high-contact location, including, but not limited to, doorknobs, toilet seats, faucets, and water fountains)."Environmental samples" can also refer to liquids (e.g., water, drinking water, fuel, petroleum separators), gases (e.g., air, medical oxygen, industrial oxygen, industrial nitrogen), soil, filters, swabs, and / or any other non-biological materials. Such environmental samples may contain biological materials, including cells and / or nucleic acids.

[0011] The term “nucleic acid” refers to polymeric forms of nucleotides of any length, including nucleic acids ranging from 2 to 100 nucleotides in length and nucleic acids exceeding 50 nucleotides in length. The term “nucleotide” refers to sugars, bases, and phosphate groups. The terms “nucleic acid base” and “base” are used interchangeably herein. The term “nucleic acid” includes polymers of canonical bases (adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)) and non-canonical bases, chemically or biochemically modified or derivatized nucleotides, and nucleotides having modified sugar-phosphate skeletons in which the conventional skeleton is replaced with a skeleton not found in nature or a synthetic skeleton. Conventional skeletons are generally considered to be the ribose-phosphate skeleton (used in ribonucleic acid (RNA)) and the deoxyribose-phosphate skeleton (used in deoxyribonucleic acid (DNA)). Skeletons not found in nature, synthetic, or other non-conventional skeletons include replacing ribose or deoxyribose with another sugar (e.g., threose), peptide, or other moiety. Examples of synthetic or other unconventional skeletons that do not exist naturally include xeno nucleic acids (XNA), peptide nucleic acids (PNA), morpholino nucleic acids, locked nucleic acids (LNA), glycol nucleic acids (GNA), 1,5-anhydrohexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), fluoroarabino nucleic acids (FANA), and threose nucleic acids (TNA). Certain nucleic acids may contain one or more nucleotides with an unconventional skeleton within a conventional skeleton—for example, one or more nucleotides may be LNA nucleotides, while the remaining nucleotides are DNA nucleotides. Nucleic acids may be of any convenient length, e.g., two or more nucleotides, e.g., four or more nucleotides, ten or more nucleotides, twenty or more nucleotides, fifty or more nucleotides, one hundred or more nucleotides, three hundred or more nucleotides, e.g., up to 500 or one thousand or more nucleotides.

[0012] As used herein, the term “isolated” means a portion of the target that is free from at least 60%, at least 75%, at least 90%, at least 95%, at least 98%, and even at least 99% of the other components to which that portion is bound prior to purification.

[0013] "Multiple" includes at least two members. In certain cases, multiple may have five or more members, for example, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, eighty or more, ninety or more, one hundred or more, three hundred or more, one thousand or more, three thousand or more, one hundred, thousand or more, one hundred, thousand or more, one hundred, thousand or more, one hundred, thousand or more, one hundred, thousand or more, one hundred, thousand or more.

[0014] A numerical range includes the number that defines the range.

[0015] The term "specific bonding" refers to the direct association between two molecules by ionic and / or hydrogen bonding interactions, including, for example, covalent interactions, electrostatic interactions, hydrophobic interactions, and interactions such as salt bridges and water bridges. Specific bonding members describe members of a molecular pair that have bonding specificity with each other. Members of a specific bonding pair may be naturally occurring or may be synthesized entirely or partially.

[0016] Specific binding members can be nucleic acids. In nucleic acids, a particular nucleotide can pair with one or more other nucleotides, for example, in canonical pairing (adenine-thymine, cytosine-guanine) or ambiguous (fluctuating) pairing (e.g., inosine-cytosine, inosine-adenine, etc.). Nucleic acids typically pair with their reverse complementary sequence, but some specific binding pairs can allow for ambiguity or mispairing. Thus, members of a pair have the property of binding specifically to each other. Such affinity or binding is generally related to its melting temperature (T). mIt can be measured by the melting temperature, and the higher the melting temperature, the stronger the bond or higher the affinity between nucleic acid pairs. "Affinity" refers to the strength of the bond, and higher binding affinity correlates with a higher melting temperature.

[0017] As used herein, the term “hybridization conditions” means the conditions under which a primer or other polynucleotide specifically hybridizes to a region of a target nucleic acid from which this primer or other polynucleotide shares a degree of complementarity. Whether a primer specifically hybridizes to a target nucleic acid depends on the degree of complementarity between the polymer and the target nucleic acid and the melting temperature (T) of the primer. MThe melting temperature is determined by factors such as the temperature at which hybridization occurs, and information can be obtained from this. The melting temperature refers to the temperature at which half of the primer-target nucleic acid duplex remains hybridized and the other half dissociates into a single strand. The Tm of the duplex can be determined experimentally or predicted using the following equation Tm = 81.5 + 16.6(log10[Na+]) + 0.41(G+C fraction) - (60 / N), (where N is the chain length and [Na+] is less than 1M). See Sambrook and Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor NY, Ch.10). Other more advanced models that depend on various parameters can also be used to predict the Tm of the primer / target duplex depending on various hybridization conditions. Approaches for achieving specific nucleic acid hybridization can be found, for example, in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, part I, chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier (1993).

[0018] As used herein, the terms “complementary” and “complementarity” refer to nucleotide sequences that non-covalently base-pair with all or part of a target nucleic acid (e.g., a region of product nucleic acid). In canonical Watson-Crick base pairing, in DNA, adenine (A) base-pairs with thymine (T), and guanine (G) base-pairs with cytosine (C). In RNA, thymine is replaced by uracil (U). Thus, A is complementary to T, and G is complementary to C. In RNA, A is complementary to U, and vice versa. Typically, “complementary” refers to nucleotide sequences that are at least partially complementary. The term “complementary” may also encompass fully complementary double helixes, where each nucleotide in one strand is complementary to each nucleotide at a corresponding position in the other strand. In certain cases, a nucleotide sequence may be partially complementary to a target, and not all nucleotides are necessarily complementary to each nucleotide in the target nucleic acid at all corresponding positions. For example, a primer may be perfectly complementary (i.e., 100%) to the target nucleic acid, or the primer and the target nucleic acid may share some degree of complementarity that is not perfect (e.g., 70%, 75%, 85%, 90%, 95%, 99%).

[0019] The percentage of identity between two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes (for example, gaps can be introduced into the sequence of the first sequence for optimal alignment). Then, the nucleotides at corresponding positions are compared, and the percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., identity % = number of identical positions / total number of positions × 100). If a position in one sequence is occupied by the same nucleotide as the corresponding position in the other sequence, then the molecules are identical at that position. A non-restrictive example of such a mathematical algorithm is described in Karlin et al., Proc.Natl.Acad.Sci.USA 90:5873-5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) described in Altschul et al., Nucleic Acids Res. 25:389-3402 (1997). When using the BLAST and Gapped BLAST programs, the default parameters for each program (e.g., NBLAST) can be used. In one embodiment, the parameters for sequence comparison can be set to score=100 and word length=12, or can be varied (e.g., word length=5 or word length=20).

[0020] As used herein, the terms “excitation maximum” and “absorption maximum” refer to the peak absorption wavelength associated with a portion. If such a portion is fluorescent (i.e., also emits photons or other radiation), “excitation maximum” and “absorption maximum” may be used interchangeably. As used herein, the term “emission maximum” refers to the peak emission wavelength associated with a fluorescent portion (e.g., a fluorophore) that emits the highest intensity radiation (e.g., light).

[0021] The methods described herein may include multiple steps. Each step may be performed after a predetermined time has elapsed between steps, if desired. Thus, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, for example, 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, the steps may be performed after incubation or a waiting period after the completion of the previous step, for example, a waiting period of several minutes to overnight.

[0022] As used herein, the terms “evaluating,” “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0023] The term “linker” or “link” refers to a linking portion that connects two groups and has a skeleton of 100 atoms or less in length. A linker or link may be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, e.g., a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms, and the linker may be linear, branched, cyclic, or single-atom. In some cases, a linker is a branched linker, referring to a linking portion that connects three or more groups. In certain cases, 1, 2, 3, 4 or 5 or more carbon atoms in the linker skeleton may be optionally substituted with heteroatoms of sulfur, nitrogen, or oxygen. In some cases, the linker skeleton may contain linking functional groups such as ethers, thioethers, aminos, amides, sulfonamides, carbamates, thiocarbamates, ureas, thioureas, esters, thioesters, or imines. The bonds between the skeletal atoms may be saturated or unsaturated, and in some cases, there may be one, two, or three or fewer unsaturated bonds in the linker skeleton. The linker may contain one or more substituents having, for example, alkyl, aryl, or alkenyl groups. The linker may, but is not limited to, polyethylene glycol; ethers, thioethers, tertiary amines, and alkyl groups that may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker skeleton may contain cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, where two or more atoms of the cyclic group, for example, two, three, or four atoms, are included in the skeleton. The linker may be cleavable or incleavable.

[0024] As used herein, the terms “water-solubilizing group,” “water-soluble group,” and “WSG” are used interchangeably and refer to a group or substituent that is well solvable in an aqueous environment, e.g., under physiological conditions, and confers improved water solubility to the molecule to which it is bound. WSGs can increase the solubility of probes, dye structures, or other components, such as donor fluorophores or acceptor fluorophores, primarily in aqueous solutions, compared to control dye structures or components lacking WSGs. A water-solubilizing group can be any convenient hydrophilic group that is well solvable in an aqueous environment.

[0025] Various water-soluble polymer groups can be adapted for use in the WSG of the target dye. Any suitable water-soluble group (WSG) can be included in the dyes described herein to provide increased water solubility. While the increase in solubility may vary, in some cases the increase (compared to compounds without WSG) is more than twofold, e.g., fivefold, tenfold, 25fold, 50fold, 100fold, or more. In some cases, the hydrophilic water-soluble group is charged, e.g., positively or negatively charged. In certain cases, the hydrophilic water-soluble group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., as described herein). In certain cases, the WSG is linear. In some embodiments, the WSG is a hydrophilic polymer, e.g., polyethylene glycol, modified PEG, peptide sequences, peptoids, carbohydrates, oxazolines, polyols, dendrons, dendritic polyglycerols, cellulose, chitosan, or derivatives thereof. The target water-soluble groups are not limited to, but include carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, hydroxyl, amines, amino acids, ammonium, guanidinium, pyridinium, polyamines and sulfonium, polyalcohols, linear or cyclic sugars, primary, secondary, tertiary or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols (including polyethers), -COOM', -SO3M', -PO3M', and -NR3+ , Y', (CH2CH2O) p R and mixtures thereof (wherein Y' can be any halogen, sulfate, sulfonate or oxygen-containing anion, p can be 1 to 500, each R can independently be H or alkyl (such as methyl), and M' can be a cationic counterion or hydrogen), --(CH2CH2O) yy CH2CH2XR yy , --(CH2CH2O) yy CH2CH2X--, --X(CH2CH2O) yy CH2CH2--, glycol and polyethylene glycol (wherein yy is selected from 1 to 1000, X is selected from O, S, and NR ZZ , and R ZZ and R YY are independently selected from H and C 1-3 alkyl). In some cases, WSG is (CH2) x (OCH2CH2) y OCH3 (wherein each x is independently an integer of 0 to 20, and each y is independently an integer of 0 to 50). In some cases, the water-solubilizing group comprises a nonionic polymer (e.g., a PEG polymer) whose terminal is substituted with an ionic group (e.g., sulfonate).

[0026] In some embodiments of the formula, the pendant group of interest comprises a substituent selected from (CH2) x (OCH2CH2) y OCH3 (wherein each x is independently an integer of 0 to 20, each y is independently an integer of 0 to 50, and benzyl optionally substituted with one or more halogen, hydroxyl, C1-C 12 alkoxy or (OCH2CH2) z OCH3 (wherein each z is independently an integer of 0 to 50)). In some instances, the substituent is (CH2)3(OCH2CH2) 11 OCH3. In some embodiments, one or more of the substituents is (CH2) x (OCH2CH2) yIt is benzyl substituted with at least one WSG group (e.g., one or two WSG groups) selected from OCH3 (wherein each x is an independent integer between 0 and 20, and each y is an independent integer between 0 and 50).

[0027] Multiple WSGs may be incorporated at a single position in the target dye via branched linkers. In certain embodiments, the branched linker is an aralkyl substituent further bisubstituted with a water-solubilizing group. Thus, in some cases, the branched linker group is a substituent of the dye that connects the dye to two or more water-solubilizing groups. In certain embodiments, the branched linker is an amino acid, for example, a lysine amino acid linked to three groups via an amino group and a carboxylic acid group. In some cases, the incorporation of multiple WSGs via branched linkers imparts a desired solubility to the dye. In some cases, the WSG is a nonionic side chain group that can impart a solubility in water greater than 50 mg / mL. In some cases, the WSG is a nonionic side chain group that can impart a solubility in water greater than 100 mg / mL. In some embodiments, the dye comprises substituents(s) selected from the group consisting of alkyl, aralkyl, and heterocyclic groups, each of which is further substituted with a water-soluble hydrophilic polymer group such as polyethylene glycol (PEG) (e.g., 6 to 24 PEG units).

[0028] Examples of water-soluble polymers that can be used in WSG include polyethylene glycol (PEG) groups or modified PEG groups. While not limited to these, the target water-soluble polymers may also include polyalkylene oxide polymers, such as polyethylene glycol "PEG" (e.g., "Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", JM Harris, Ed., Plenum Press, New York, NY (1992); and "Poly(ethylene glycol) Chemistry and Biological Applications", JM Harris and S. Zalipsky, Eds., ACS (1997); and international patent applications: WO90 / 13540, WO92 / 00748, WO92 / 16555, WO94 / 04193, WO94 / 14758, WO94 / 17039, WO94 / 18247, WO94 / 28937, WO95 / 11924, WO96 / 00080, WO96 / 23794 Nos. WO98 / 07713, WO98 / 41562, WO98 / 48837, WO99 / 30727, WO99 / 32134, WO99 / 33483, WO99 / 53951, WO01 / 26692, WO95 / 13312, WO96 / 21469, WO97 / 03106, WO99 / 45964, and U.S. Patent No. 4,179,337; No. 5 ,075,046;No.5,089,261;No.5,100,992;No.5,134,192;No.5,166,309;No.5,171,264;No.5,213,891;No. No. 5,219,564; No. 5,275,838; No. 5,281,698; No. 5,298,643; No. 5,312,808; No. 5,321,095; No. 5,324,844; No. No. 5,349,001; No. 5,352,756; No. 5,405,877; No. 5,455,027; No. 5,446,090; No. 5,470,829; No. 5,478,805; No. 5,567,422; No. 5,605,976; No. 5,612,460; No. 5,614,549; No. 5,618,528; No. 5,672,662; No. 5,637,749;No. 5,643,575; No. 5,650,388; No. 5,681,567; No. 5,686,110; No. 5,730,990; No. 5,739,208; No. 5,756,593; No. 5,808,096 ;No. 5,824,778;No. 5,824,784;No. 5,840,900;No. 5,874,500;No. 5,880,131;No. 5,900,461;No. 5,902,588;No. 5,919,442 See issues 5,919,455, 5,932,462, 5,965,119, 5,965,566, 5,985,263, 5,990,237, 6,011,042, 6,013,283, 6,077,939, 6,113,906, 6,127,355, 6,177,087, 6,180,095, 6,194,580, and 6,214,966.

[0029] Examples of the target water-soluble polymers include, but are not limited to, polyalkylene oxides, polyamide alkylene oxides, or derivatives thereof, such as those containing polyalkylene oxides and polyamide alkylene oxides with the ethylene oxide repeating unit of formula -(CH2-CH2-O)-. Further examples of the target polymers include those of the formula -[C(O)-XC(O)-NH-Y-NH]n- or -[NH-Y-NH-C(O)-XC(O)] n -Includes polyamides having a molecular weight greater than 1,000 Daltons (wherein X and Y are divalent groups which may be the same or different, which may be branched or linear, n is a discrete integer between 2 and 100, e.g., 2 to 50, and either or both of X and Y contain biocompatible, substantially non-antigenic, water-soluble repeating units which may be linear or branched). Further examples of water-soluble repeating units include ethylene oxide of the formula -(CH2-CH2-O)- or -(O-CH2-CH2)-. The number of such water-soluble repeating units may vary significantly, and the number of such units may be 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6 to 100, e.g., 2 to 50 or 6 to 50. An example of an embodiment is where one or both of X and Y are -((CH2)n1 -(CH2-CH2-O) n2 -(CH2)- or -((CH2) n1 -(O-CH2-CH2) n2 -(CH2) n-1 -)(wherein n1 is selected from 1-6, 1-5, 1-4, or 1-3, and n2 is selected from 2-50, 2-25, 2-15, 2-10, 2-8, or 2-5). A further example of one embodiment is where X is -(CH2-CH2)- and Y is -(CH2-(CH2-CH2-O)3-CH2-CH2-CH2)- or -(CH2-CH2-CH2-(O-CH2-CH2)3-CH2)-.

[0030] The term modified polymer, such as modified PEG, refers to a water-soluble polymer that has been modified or derivatized at one or both ends to include terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy, or substituted alkoxy) and / or terminal linking functional groups (e.g., amino or carboxylic acid groups suitable for linking via amide bond formation) that are suitable for linking the polymer to a target molecule (e.g., to a light-harvesting chromophore via branching groups). The target water-soluble polymer can be adapted to include any convenient linking groups. In some cases, it will be understood that water-soluble polymers may exhibit some dispersibility with respect to polymer length, depending on the method of preparation and / or purification of the polymer starting materials. In some cases, water-soluble polymers are monodisperse.

[0031] Water-soluble polymers may contain one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties containing one or more repeating units, diamino and / or diacid units, natural or unnatural amino acids or their derivatives, and aliphatic moieties containing alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., which may contain, for example, up to 18 carbon atoms, or even further polymer chains.

[0032] The water-soluble polymer moiety, or one or more spacers or linkers of the polymer moiety if present, may contain biostable or biodegradable polymer chains or units. For example, polymers with repeating links have varying degrees of stability under physiological conditions depending on the instability of the linkage. Polymers with such links can be classified, for example, from less stable to more stable, based on their relative hydrolysis rates under physiological conditions, which are derived from the known hydrolysis rates of low molecular weight analogs, into categories such as polyurethane (-NH-C(O)-O-) > polyorthoester (-OC((OR)(R'))-O-) > polyamide (-C(O)-NH-). Similarly, the linking systems for attaching water-soluble polymers to target molecules can be biostable or biodegradable, for example, from least stable to most stable: carbonate (-OC(O)-O-) > ester (-C(O)-O-) > urethane (-NH-C(O)-O-) > orthoester (-OC((OR)(R'))-O-) > amide (-C(O)-NH-). In general, depending on the instability of the sulfate group, it may be desirable to avoid the use of sulfated polysaccharides. Furthermore, the use of polycarbonates and polyesters may be less desirable. These linkages are provided as examples and are not intended to limit the types of linkages that can be used in polymer chains or linking systems of water-soluble polymers useful in WSG disclosed herein.

[0033] Water-soluble groups (WSGs) can impart solubility in water of greater than 10 mg / mL, for example, greater than 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL to the target probe or dye structure. In specific cases, branched nonionic water-soluble groups (WSGs) can impart solubility in water (e.g., aqueous buffer) of 20 mg / mL or more, for example, greater than 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more to the target probe or dye structure. It will be understood that water-soluble dipyromethene dyes can form separated water-solvable nanoparticles in aqueous systems under certain conditions. In certain cases, water-solvated nanoparticles are resistant to aggregation and have been found to be useful in a variety of biological assays.

[0034] The terms "polyethylene oxide," "PEO," "polyethylene glycol," and "PEG" are used interchangeably, and the formula is (CH2----O--). nThis refers to a polymer group comprising a chain or derivative thereof represented by -. In some embodiments, "n" is 5000 or less, e.g., 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, e.g., 3 to 15, or 10 to 15. The PEG polymer group may be of any convenient length and may include various terminal groups and / or further substituents, e.g., but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide terminal groups and / or substituents. Examples of PEG groups suitable for use in target multichromophores include those described in "Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates" by S. Zalipsky, Bioconjugate Chemistry 1995, 6(2), 150-165; and "Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy" by Zhu et al., Chem. Rev., 2012, 112(8), pp 4687-4735.

[0035] The term "alkyl" refers to a saturated, branched, or linear monovalent hydrocarbon group derived by removing one hydrogen atom from a single carbon atom of a parent alkane, either by itself or as part of another substituent. Examples of alkyl groups of interest include, but are not limited to, methyl; ethyl; propyl, e.g., propan-1-yl or propan-2-yl; and butyl, e.g., butane-1-yl, butane-2-yl, 2-methyl-propan-1-yl, or 2-methyl-propan-2-yl. In some embodiments, the alkyl group contains 1 to 20 carbon atoms. In some embodiments, the alkyl group contains 1 to 10 carbon atoms. In certain embodiments, the lower alkyl group contains 1 to 6 carbon atoms, e.g., 1 to 4 carbon atoms. This term includes, for example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0036] The term "substituted alkyl" refers to a group where one or more carbon atoms in the alkyl chain are -O-, -N-, -S-, -S(O) n- (wherein n is 0 to 2), -NR- (wherein R is hydrogen or alkyl), etc., are optionally substituted with heteroatoms, and include alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NR a R b (In the formula, R a and R b This refers to an alkyl group as defined herein, having 1 to 5 substituents selected from the group consisting of hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle, which may be the same or different.

[0037] "Alkoxy" refers to an -O-alkyl group, where alkyl is as defined herein. Examples of alkoxys include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0038] The term "substituted alkoxy" refers to substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- groups, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0039] "Alkenyl" refers to a branched or linear, cyclic or acyclic hydrocarbonyl group of a monoradical containing a carbon-carbon double bond. Exemplary alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicocenyl, and tetracocenyl. In some cases, alkenyl groups contain 1 to 24 carbon atoms, for example, 1 to 18 or 1 to 12 carbon atoms. The term "lower alkenyl" refers to an alkenyl group having 1 to 6 carbon atoms.

[0040] "Alkynyl" or "alkyne" refers to a linear or branched monovalent hydrocarbyl group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and at least 1, preferably 1 to 2 triple-bond unsaturated sites. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CH2C≡CH).

[0041] The terms "substituted alkynyl" or "substituted alkyne" refer to an alkynyl group as defined herein, having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0042] A "heterocyclyl" refers to a non-aromatic monoradical cyclic group that contains a heteroatom (e.g., O, S, N) as a ring atom (i.e., distinguishing heterocyclyl groups from heteroaryl groups). Examples of heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0043] "Amino" refers to the -NH2 group. The term "substituted amino" refers to the group -NRR, where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, except that at least one R is not hydrogen.

[0044] The term "aryl" refers to a monovalent aromatic hydrocarbon group derived by removing one hydrogen atom from a single carbon atom in an aromatic ring system, either by itself or as part of another substituent. The aryl groups of interest include, but are not limited to, groups derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluorantene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indan, indene, naphthalene, octacene, octafene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentafene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, etc. In certain embodiments, the aryl group contains 6 to 20 carbon atoms. In certain embodiments, the aryl group contains 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0045] Unless otherwise specifically restricted by the definition of aryl substituents, "substituted aryl" refers to an aryl group substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkalyl, aryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0046] A "heteroaryl" refers to a monovalent heteroaromatic group derived by removing one hydrogen atom from a single atom of a heteroaromatic ring system, either by itself or as part of another substituent. The target heteroaryl group is not limited to groups derived from acridine, arsindol, carbazole, β-carbolin, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indidine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthoridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolidine, quinoxaline, tetrazoline, thiadiazole, thiazoline, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole, etc. In specific embodiments, the heteroaryl group is derived from a 5-20 member heteroaryl. In certain embodiments, the heteroaryl group is derived from a 5- to 10-membered heteroaryl group. In certain embodiments, the heteroaryl group is derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0047] "Heterocyclic," "heterocyclic," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having monocyclic or multi-fused rings, including condensation, bridging, and spirocyclic systems, and containing 3 to 20 ring atoms including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, and in fused ring systems, one or more of the rings may be cycloalkyl, aryl, or heteroaryl, provided that the bond site passes through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms(s) of the heterocyclic group are selectively oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0048] Examples of heterocyclic and heteroaryl compounds include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indidine, isoindole, indole, dihydroindole, indazole, purine, quinoridine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carborin, phenanthridine, acridine, phenanthroline, isothiazole, phena Examples include din, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinil, thiomorpholinil (also called thiamorpholinil), 1,1-dioxothiomorpholinil, piperidinil, pyrrolidine, and tetrahydrofuranil.

[0049] Unless otherwise specifically restricted by the definition of substituents, "substituted heteroaryl" refers to a heteroaryl group substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0050] The terms "alkalyl" or "aralkyl" refer to alkylene, substituted alkylene, and aryl groups, as defined herein, specifically the alkylene-aryl and substituted alkylene-aryl groups.

[0051] The alkylene is preferably either linear or branched, and is -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 This term refers to a divalent aliphatic hydrocarbyl group having preferably 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, which is optionally interrupted by one or more groups selected from the following. Examples of this term include methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), etc. "Substituting alkylene" refers to an alkylene group in which 1 to 3 hydrogen atoms are substituted with substituents, as described for carbon in the definition of "substitution" below.

[0052] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by the same or different substituent(s). The substituent(s) of interest are not limited to alkylenedioxy (such as methylenedioxy), -M, and -R. 60 , -O - ,=O,-OR 60 , -SR 60 , -S - ,=S,-NR 60 R 61 ,=NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O - -S(O)2OH, -S(O)2R 60 -OS(O)2O - -OS(O)2R 60 ,-P(O)(O - )2, -P(O)(OR 60 )(O- ), -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 ,-C(S)R 60 , -C(O)OR 60 -C(O)NR 60 R 61 ,-C(O)O - , -C(S)OR 60 , -NR 62 C(O)NR 60 R 61 , -NR 62 C(S)NR 60 R 61 , -NR 62 C(NR 63 )NR 60 R 61 and -C(NR 62 )NR 60 R 61 (In the formula, M is a halogen, and R 60 , R 61 , R 62 and R 63 Independently, is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or optionally, R 60 and R 61 They, together with the nitrogen atom to which they are bonded, form a cycloheteralkyl or substituted cycloheteralkyl ring; R 64 and R 65 Independently, is hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or optionally, R 64 and R 65 These include (which, together with the nitrogen atom to which they are bonded, form a cycloheteroalkyl or substituted cycloheteroalkyl ring). In certain embodiments, substituents include -M, -R 60 ,=O,-OR 60 , -SR60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2R 60 , -OS(O)2O - , -OS(O)2R 60 , -P(O)(O - )2, -P(O)(OR 60 )(O - ), -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(S)R 60 , -C(O)OR 60 , -C(O)NR 60 R 61 , -C(O)O - , -NR 62 C(O)NR 60 R 61 are included. In certain embodiments, the substituents include -M, -R 60 , =O, -OR 60 , -SR 60 , -NR 60 R 61 , -CF3, -CN, -NO2, -S(O)2R 60 , -P(O)(OR 60 )(O - ), -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(O)OR 60 , -C(O)NR 60 R 61 , -C(O)O - are included. In certain embodiments, the substituents include -M, -R 60 , =O, -OR 60 , -SR 60 , -NR 60 R 61 , -CF3, -CN, -NO2, -S(O)2R 60 , -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(O)OR 60 , -C(O)O- R 60 , R 61 and R 62 The definition is as described above. For example, a substituent may have one, two, or three substituents selected from methylenedioxy substituents, halogen atoms, (1-4C)alkyl groups, and (1-4C)alkoxy groups. When the substituted group is an aryl or heteroaryl group, the substituent(s) (e.g., those described herein) may be referred to as “aryl substituent(s)”.

[0053] It will be understood that polymers obtained by defining substituents having further substituents on themselves for all substituents defined above (for example, substituted aryls having a substituted aryl group as a substituent, where the substituent itself is substituted with a substituted aryl group, and that substituent is further substituted with a substituted aryl group) are not intended to be included herein. In such cases, the maximum number of such substitutions is three. For example, the sequential substitution of substituted aryl groups specifically intended herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0054] "Acyl" refers to a group of the formula -C(O)R where R is alkyl, alkenyl, or alkynyl. For example, the acetyl group has the formula -C(O)CH3.

[0055] "Halo" and "halogen" refer to chloro, bromo, fluoro, and iodo groups.

[0056] "Carboxyl," "carboxy," and "carboxylate" refer to the -CO2H group and their salts.

[0057] "Sulfonyl" refers to the group -SO2R (wherein R is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and their substituted forms). Exemplary sulfonyl groups include -SO2CH3 and -SO2(C6H5).

[0058] Unless otherwise specified, a reference to an atom includes all isotopes of that atom. For example, a reference to H means, 1 H, 2 H (i.e., D) and 3 This means that H (i.e., T) is included, and the reference to C is, 12 C and all isotopes of carbon ( 13 This means that it includes (such as C). Furthermore, any group listed includes all stereoisomers of that group.

[0059] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is obtained by naming the terminal portion of the functional group, followed by the functional groups adjacent to the bond site. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0060] With respect to any of the groups disclosed herein that contain one or more substituents, it will be understood that such groups do not contain any substitutions or substitution patterns that are sterically impractical and / or synthetically unfeasible. Furthermore, the compounds of interest include all stereochemical isomers resulting from the substitutions of these compounds. [Modes for carrying out the invention]

[0061] Scaffolded chromophores for nucleic acid detection, as well as methods and uses thereof, are provided. The scaffolded chromophores of embodiments of the present invention include: a probe formed from a nucleic acid complementary to a target sequence; a dye structure ligated to the first end of the nucleic acid, comprising a non-conjugated polymer backbone, one or more excitable donor fluorophores ligated to the non-conjugated polymer backbone, and one or more acceptor fluorophores ligated to the non-conjugated polymer backbone, which are in an energy transfer relationship with the donor fluorophores; and a quencher ligated to the second end of the nucleic acid, which are in an energy transfer relationship with one or more acceptor fluorophores. Furthermore, methods for preparing and using scaffolded chromophores for nucleic acid detection, as well as kits comprising dyes, which are found to be used in embodiments of the present method, are provided.

[0062] Before describing exemplary embodiments in more detail, the following definitions are provided to illustrate and define the meaning and scope of terms used in the description.

[0063] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art in which the present invention pertains. Furthermore, for clarity and ease of reference, certain terms are defined below. In addition, any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but representative exemplary methods and materials are described herein.

[0064] Where a range of values ​​is provided, it is understood that each value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, unless the context clearly indicates otherwise, and any other stated or between values ​​within that stated range are included in the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range, subject to any specifically excluded limits within the stated range, and are also included in the invention. If a stated range includes one or both limits, the range excluding one or both of those limits is also included in the invention.

[0065] In this specification, certain ranges are presented with numbers preceded by the term “approximately.” The term “approximately” is used herein to provide literal support for the exact number preceded by the term, as well as for numbers that are close to or approximate the number preceded by the term. In determining whether a number is close to or approximates a specifically stated number, the close or approximate unspecified number may, in the context in which it is presented, provide a substantial equivalent of the specifically stated number.

[0066] All publications and patents referenced herein are incorporated herein by reference in such a way that it specifically and individually indicates that each individual publication or patent is incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials to which the publications are referenced. Any reference to a publication is for its disclosure prior to the filing date and should not be construed as an acknowledgment that the present invention has no prior rights to such publication by prior art. Furthermore, the publication dates presented may differ from the actual publication dates and may need to be independently verified.

[0067] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as a prior art for the use of exclusive terms such as “exclusively” and “only” in relation to the description of elements of a claim or the use of “negative” limitations.

[0068] As will be obvious to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated or combined with features of any of several other embodiments without departing from the scope or spirit of the invention. Any method described may be performed in the order of events described or in any other logically possible order.

[0069] Apparatus and methods are described, or will be described, with functional descriptions to improve the flow of the text, but claims should never be interpreted as being necessarily limited by constructing a limitation of “means” or “steps” unless explicitly formulated under Section 112 of the United States Patent Act, and the meaning of the definitions and the full scope of equivalents provided by the claims should be given under the doctrine of equivalents, and if the claims are explicitly formulated under Section 112 of the United States Patent Act, it should be clearly understood that the full statutory equivalents should be given under Section 112 of the United States Patent Act.

[0070] In further describing specific embodiments of the present invention, we will first examine in more detail the scaffolded chromophore for nucleic acid detection, followed by an examination of methods for using and preparing the dye, and an examination of a kit containing the scaffolded chromophore for nucleic acid detection.

[0071] Scaffolded chromophores for nucleic acid detection As summarized above, this disclosure provides scaffolded chromophores for use in nucleic acid detection. As used herein, “scaffolded chromophore” refers to a chromophore containing fluorophores arranged in an energy transfer relationship such as FRET. Scaffolded chromophores include donor and acceptor chromophores arranged on a common structure such as a skeleton. Detection can be qualitative (e.g., presence / absence), quantitative (e.g., amount of target present), and / or semi-quantitative (e.g., relative amount of target). Scaffolded chromophores can be used as probes in nucleic acid amplification reactions, including polymerase chain reactions (PCR) and their variations, such as quantitative PCR (qPCR), digital PCR (dPCR), and other related forms of PCR. Further amplification methods include isothermal amplification reactions, such as nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand-displacement amplification (SDA), recombinase polymerase amplification (RPA), and rolling circle amplification (RCA).

[0072] As further summarized above and shown in Figure 1, a particular embodiment relates to a probe for identifying a target nucleic acid. To detect the target nucleic acid, the particular embodiment includes a nucleic acid 102 complementary to the target sequence. In the particular embodiment, the nucleic acid is attached to a dye structure 104 and a quencher 106. Further details of each component are discussed further in the text below.

[0073] nucleic acid Nucleic acid 102 is composed of multiple nucleotides that form a chain from the 5' end to the 3' end. Nucleic acid 102 can be of any length sufficient to hybridize to a specific target sequence and / or be specific to a specific target sequence under specific conditions, such as reaction conditions for amplification reactions. The temperature that enables hybridization with the target sequence is the melting temperature (or T) of the particular sequence. m ) can be measured by T mThis can generally be measured and determined using a variety of available methods. In certain embodiments, nucleic acid 102 may be of any convenient length, e.g., two or more nucleotides, e.g., four or more nucleotides, ten or more nucleotides, twenty or more nucleotides, fifty or more nucleotides, one hundred or more nucleotides, three hundred or more nucleotides, e.g., up to five hundred or one thousand or more nucleotides. Some embodiments include nucleic acids having 10 to 50 nucleotides. Certain embodiments include nucleic acids having 18 to 30 nucleotides.

[0074] Nucleic acids 102 and / or individual nucleotides are formed from canonical bases (adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)) and non-canonical bases (also called analogs), chemically or biochemically modified or derivatized nucleotides, and nucleotides having modified sugar-phosphate skeletons in which the conventional skeleton is replaced with a skeleton that does not exist in nature or a synthetic skeleton.

[0075] Various nucleotide modifications and analogues are known in the art, including 1-methyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, 2-methyladenosine, 2-O-ribosylphosphateadenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, and N6,N6-dimethyladenosine. Nosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxynorvalylcarbamoyladenosine, 1,2-O-dimethyladenosine, N6,2-O-dimethyladenosine, 2-O-methyladenosine, N6,N6,O-2-trimethyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, 2 - Thiocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5-formyl-2-O-methylcytidine, 5,2-O-dimethylcytidine, 2-O-methylcytidine, N4,2-O-dimethylcytidine, N4,N4,2-O-trimethylcytidine, 1-methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2-O-ribo Sylphosphate guanosine, 7-methylguanosine, low-modified hydroxywybutosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2,N2-dimethylguanosine, 4-demethylwyosine, epoxyquosine, hydroxywybutosine, isowiosine, N2,7,2-O-trimethylguanosine, N2,2-O-dimethylguanosine, 1,2-O-dimethylguanosine, 2-O-methylguanosine, N2,N2,2-O-trimethylguanosine, N2,N2,7-Trimethylguanosine, Peroxywybutosin, Galactosyl-Quosin, Mannosyl-Quosin, Quosin, Archeosin, Wybutosin, Methylwyosin, Wyosin, 2-Thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-Methyluridine, 4-Thiouridine, 5-Methyl-2-Thiouridine, 5-Methylaminomethyluridine, 5-Carboxymethyluridine, 5-Carboxymethylaminomethyluridine, 5-Hydroxyuridine 5-methyluridine, 5-taurinomethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 3,2-O-dimethyluridine, 5-carboxymethylaminomethyl-2-O -Methyluridine, 5-Carbamoylmethyl-2-O-methyluridine, 5-Methoxycarbonylmethyl-2-O-methyluridine, 5-(Isopentenylaminomethyl)-2-O-methyluridine, 5,2-O-dimethyluridine, 2-O-methyluridine, 2-thio-2-O-methyluridine, Uridine 5-oxyacetic acid, 5-Methoxycarbonylmethyluridine, Uridine 5-oxyacetic acid methyl ester, 5-Methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxy This includes methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)psoidouridine, 1-methylpsoidouridine, 3-methylpsoidouridine, 2-O-methylpsoidouridine, inosine, 1-methylinosine, 1,2-O-dimethylinosine, and 2-O-methylinosine. Each of these may be a component of the nucleic acids of this disclosure.

[0076] Conventional skeletons are generally considered to be the ribose-phosphate skeleton (used in ribonucleic acid (RNA)) and the deoxyribose-phosphate skeleton (used in deoxyribonucleic acid (DNA)). Synthetic or other unconventional skeletons that do not exist naturally involve replacing ribose or deoxyribose with another sugar (e.g., threose), a peptide, or other moiety. Examples of synthetic or other unconventional skeletons that do not exist naturally include xeno nucleic acids (XNA), peptide nucleic acids (PNA), morpholino nucleic acids, locked nucleic acids (LNA), glycol nucleic acids (GNA), 1,5-anhydrohexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), fluoroarabino nucleic acids (FANA), and threose nucleic acids (TNA). Certain nucleic acids may contain one or more nucleotides with an unconventional skeleton within a conventional skeleton—for example, one or more nucleotides may be LNA nucleotides, while the remaining nucleotides are DNA nucleotides.

[0077] pigment structure The dye structure 104 can be constructed as a scaffolded chromophore. In certain embodiments, the dye structure 104 comprises a polymer backbone 108, one or more donor fluorophores 110, and one or more acceptor fluorophores 112. The fluorophores of the donor 110 and acceptor 112 are bonded to the polymer backbone 108. In certain embodiments, the polymer backbone is non-conjugated. In certain embodiments, the dye structure 104 is bonded to the end (e.g., 5' end or 3' end) of the nucleic acid 102. In certain embodiments, the dye structure 104 is bonded to the 5' end of the nucleic acid 102.

[0078] Polymer skeleton As summarized above, the dye structure of the embodiment includes a polymer backbone 108. In some cases, the polymer backbone 108 is composed of non-conjugated repeating units having any convenient configuration, such as linear, branched, or dendrimer configurations. The polymer backbone 108 may be a linear polymer. The polymer backbone 108 may be branched. In some cases, the dye structure includes a plurality of pendant donor chromophore groups (e.g., fluorophores of donor 110 and acceptor 112) independently linked to the non-conjugated repeating units of the polymer backbone. The configuration of the pendant groups can be attached during or after the synthesis of the polymer backbone. The incorporation of the pendant groups can be achieved in a sequence-specific manner by random configuration, block configuration, or stepwise synthesis, depending on the specific synthesis method used.

[0079] The term "unit" refers to a structural subunit of a polymer. The term "unit" includes monomers, comonomers, coblocks, repeating units, and so on. A "repeating unit" or "repeat unit" is a subunit of a polymer defined by the minimum number of distinct structural features required for a unit to be considered a monomer; therefore, when a unit is repeated n times, the resulting structure represents that polymer or block. In some cases, a polymer may contain two or more distinct repeating units; for example, if the polymer is a multiblock polymer, a random arrangement of units, or a defined arrangement, each block may define a distinct repeating unit. Various arrangements of repeating units or blocks are possible, and it will be understood that in the illustrated formulas of polymer backbones described herein, any convenient linear arrangement of various lengths may be included within the overall structure of the polymer. A polymer may also be represented by a formula relating to the molar percentage value of each unit in the polymer, and it will be understood that such a formula may represent various arrangements of repeating units, such as random polymers, multiblock polymers, or defined arrangements of residues. In some cases, the repeating units of a polymer contain a single monomer group. In certain cases, the repeating units of a polymer contain two or more monomer groups, i.e., comonomer groups, for example, two, three, four or more comonomer groups. The terms “comonomer” or “comonomer group” refer to a polymer structural unit that may itself be part of the repeating units of the polymer.

[0080] The polymer backbone 108 of the dye structure 104 can have any convenient length. In some cases, the specific number of monomer repeating units or segments of the chromophore may be within the range of 2 to 500,000, e.g., 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or e.g., 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments. In some cases, the specific number of monomer repeating units or segments in the skeleton may be within the range of 2 to 1,000, e.g., 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units or segments. In certain cases, the specific number of monomer repeating units or segments in the skeleton may be within the range of 2 to 500, e.g., 2 to 400, 2 to 300, 2 to 200, or 2 to 100 units or segments. In certain cases, the specific number of monomer repeating units or segments in the skeleton may be within the range of 2 to 100 repeating units, e.g., 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 units or segments.

[0081] The polymer backbone 108 may have a random configuration of non-conjugated repeating units. The polymer backbone 108 may include block or coblock configurations of non-conjugated repeating units. Alternatively, the polymer backbone may include a specific defined sequence of non-conjugated repeating units, e.g., amino acid residues of a polypeptide sequence. These configurations can be characterized by polymer segments of repeating units (e.g., those described herein), which themselves can be repeated throughout the modular scaffold.

[0082] "Non-conjugated" means that at least a portion of the repeating units contain saturated skeletal groups (e.g., groups with two or more consecutive single covalent bonds) that prevent π-conjugation or extended delocalized electronic structures along the polymer backbone from one repeating unit to the next. Even if one repeating unit is not conjugated to an adjacent repeating unit, such a repeating unit may contain one or more isolated unsaturated groups (e.g., alkenylene or alkynylene groups) and / or aryl or heteroaryl groups containing unsaturated bonds, and these groups may be part of the backbone. In some cases, each repeating unit of the polymer backbone contains a side chain containing a linking pendant group or a chemoselective tag for linking to a pendant group.

[0083] In certain embodiments of the dye structure 104, the polymer backbone 108 is a linear polymer. In certain cases, the linear polymer is selected from peptides, peptoids, hydrocarbon polymers, PEG polymers, carbohydrates, lipids, and polynucleotides. In certain cases, the linear polymer is a peptide. In certain cases, the linear polymer is a peptoid. In certain cases, the polymer is a hydrocarbon polymer. In certain other cases, the polymer is a PEG polymer. Further details regarding the polymer backbone that may be used in embodiments of the present invention can be found in PCT application number PCT / US2019 / 024662, published as International Publication No. 2019 / 191482, and PCT application number PCT / US2020 / 019510, published as International Publication No. 2020 / 222894, the disclosures of which are incorporated herein by reference.

[0084] In certain cases, the pigment structure contains a linear peptide backbone of 2 to 100 amino acids, e.g., 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 amino acids. In some cases, the linear peptide backbone contains 2 or more amino acids, e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, up to 100 amino acids. In certain cases, the pigment structure contains a linear peptide backbone of 5 to 30 amino acids, e.g., 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids.

[0085] If desired, the polymer skeleton present in the dye structure of the embodiment of the present invention may be substituted with one or more water-soluble groups (WSGs), for example, as defined above.

[0086] Donor and acceptor fluorophores As provided above, the dye structure of a particular embodiment may include one or more donor fluorophores 110 and one or more acceptor fluorophores 112, each bonded to the polymer backbone 108. In a particular embodiment, the donor 110 and acceptor 112 fluorophores are in an energy transfer relationship. An energy transfer relationship is a situation in which emission from the donor fluorophore 110 is absorbed by the acceptor fluorophore 112. In a particular case, the emission maximum from the donor fluorophore 110 is the excitation (or absorption) maximum of the acceptor fluorophore 112. The number of donor fluorophores 110 may be 1 to 20 fluorophores, e.g., 1 to 20, 1 to 15, 1 to 10, 1 to 5, 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any integer fluorophore between 1 and 20. Furthermore, the number of acceptor fluorophores can be 1 to 20 fluorophores, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or any integer fluorophore between 1 and 20.

[0087] By allowing various combinations or numbers of donor and acceptor fluorophores, certain embodiments enable a tunable fluorescence mechanism for nucleic acid detection. Such tunability can be achieved by changing the amounts of donor and / or acceptor fluorophores within the dye structure. In some cases, the emission from acceptor fluorophores is proportional to the number of donor fluorophores, allowing for greater emission as the number of donor fluorophores increases. Such a process can be used to increase the sensitivity of the probe, for example, to enable the detection of minute or minute amounts of target sequences. For example, to increase the emission from acceptor fluorophores, the probe may contain more donor fluorophores, or even more than the number of acceptor fluorophores.

[0088] The combination of donor fluorophore and acceptor fluorophore can be selected from known fluorophore having a FRET relationship. In certain cases, the excitation maximum of the donor fluorophore is in the range of approximately 300 nm to approximately 700 nm. Such wavelengths include near ultraviolet (near UV) to red wavelengths. In certain cases, the donor fluorophore may have excitation maxima in the range of near UV (approximately 300 nm to approximately 380 nm), violet (approximately 380 nm to approximately 440 nm), blue (approximately 440 nm to approximately 485 nm), cyan (approximately 485 nm to approximately 510 nm), green (approximately 510 nm to approximately 565 nm), yellow (approximately 565 nm to approximately 590 nm), orange (approximately 590 nm to approximately 625 nm), or red (approximately 625 nm to approximately 700 nm). Generally, the emission maximum is at a higher wavelength than the excitation maximum, and the emission maximum obtained for a donor fluorophore according to a particular embodiment may be 380–440 nm, 440–485 nm, 485–510 nm, 510–565 nm, 565–590 nm, 590–625 nm, or 625–725 nm, depending on the excitation and Stokes shift of the particular fluorophore. Exemplary chromophores include bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores. Such fluorophores are listed in U.S. Patent Nos. 11,702,547, 11,643,556, 11,485,825, 11,214,688, 11,209,438, 10,851,212, 10,844,228, 10,703,864, 10,663,476, 10,648,900, 10,240,004, 10,073,093, 10,018,640, and 9. The disclosures are described in one or more of the following: U.S. Patent Application Publications Nos. 797,899 and 9,758,625; U.S. Patent Application Publications Nos. 2023 / 0152327, 2022 / 0348770, 2021 / 0032474 and 2020 / 0225143; and U.S. Patent Applications Nos. 18 / 201,940, 18 / 201,649 and 18 / 120,554; the disclosures thereof are incorporated herein by reference in their entirety.

[0089] As described above, FRET utilizes an acceptor fluorophore having an excitation distribution that includes the emission maximum of a selected donor fluorophore. In certain embodiments, the emission maximum of the selected donor fluorophore is approximately equal to the excitation maximum of the acceptor fluorophore (e.g., ±25 nm). In certain embodiments, one or more acceptor fluorophores have excitation maxima in the range of 450 to 750 nm. Such maxima may include excitation maxima in the blue (approx. 440 nm to approx. 485 nm), blue-green (approx. 485 nm to approx. 510 nm), green (approx. 510 nm to approx. 565 nm), yellow (approx. 565 nm to approx. 590 nm), orange (approx. 590 nm to approx. 625 nm), or red (approx. 625 nm to approx. 740 nm) regions, depending on a particular type of acceptor fluorophore, the desired emission maximum, and its Stokes shift. Considering excitation and Stokes shift, various acceptor fluorophores can have emission maxima in the range of approximately 500 nm to 875 nm, which generally falls within the blue-green to near-infrared (near-IR) light range, for example, 475 nm to 600 nm, 600 nm to 700 nm, or 700 nm to 800 nm.

[0090] These acceptor fluorophores can be selected from rhodamine, perylene, diimide, coumarin, xanthene, cyanine, polymethine, pyrene, thiazine, acridine, dipyrometheneboronedifluoride, naphthalimide, phycobiliprotein, peridine (peridinin) chlorophyll protein, their conjugates, and combinations thereof. In certain embodiments, acceptor fluorophore (A) is a cyanine dye, xanthene dye, coumarin dye, thiazine dye, or acridine dye. In some cases, the acceptor fluorophore (A) is selected from DY431, DY485XL, DY610, DY640, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY778, DY782, DY800, DY831, and diethylaminocoumarin. The target fluorescent dyes include, but are not limited to, Texas Red, Cascade Blue, Cascade Yellow, Coumarin, Cy5, Cy5.5, Cy-Chrome, Phycoerythrin, PerCP (Peridinin-chlorophyll-α protein), PerCP-Cy5.5, Lucifer Yellow, Marina Blue, Oregon Green 488, Alexa Fluor350, Alexa Fluor430, Alexa Fluor488, Alexa Fluor594, Alexa Fluor633, Alexa Fluor647, Alexa Fluor660, Alexa Fluor680, Alexa Fluor700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY576 / 589, BODIPY581 / 591, BODIPY630 / 650, BODIPY650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, their conjugates, and combinations thereof. While not limited to specific examples, the target lanthanide chelates include europium chelate, terbium chelate, and samarium chelate.In some embodiments, the polymer dye structure includes a multichromophore linked to an acceptor fluorophore selected from Cy5, Cy5.5, Cy7, Alexa488, Alexa647, and Alexa700. In certain embodiments, the polymer dye structure includes a multichromophore linked to an acceptor fluorophore selected from a Dyomics dye (such as DY431, DY610, DY633, DY640, DY651, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY754, DY778, DY782, DY800, or DY831) and diethylaminocoumarin. In certain cases, the acceptor fluorophore (A) is used in Texas Red, California Red, iFluor594, Cascade Blue, Cascade Yellow, Coumarin, Cy5(registered trademark), Cy5.5(registered trademark), Cy7(registered trademark), Cy-Chrome, DyLight350, DyLight405, DyLight488, DyLight594, DyLight633, DyLight649, DyLight680, DyLight750, DyLight800, Phycoerythrin, PerCP (Peridinin-chlorophyll-α protein), PerCP-Cy5.5, Lucifer Yellow, Marina Blue, Oregon Green 488, Alexa Fluor(registered trademark) 350, Alexa Fluor(registered trademark) 430, Alexa Fluor(registered trademark) 488, Alexa Fluor(registered trademark) 594, Alexa Fluor(registered trademark) 633, Alexa Fluor(registered trademark) 647, and Alexa Fluor(registered trademark) 694. Selected from Fluor(registered trademark) 660, Alexa Fluor(registered trademark) 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY(registered trademark) 576 / 589, BODIPY(registered trademark) 581 / 591, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) R6G, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, their conjugates, and combinations thereof.Various dyes are known in the art and / or U.S. Patents No. 11,668,716, No. 11,624,089, No. 11,407,773, No. 11,371,078, No. 11,214,688, No. 11,130,865, No. 11,119,107, No. 10,605,813, and No. 10,228,37 No. 5, No. 10,053,484, No. 9,863,947, No. 9,797,899, No. 9,719,998, No. 9,551,029, No. 9,493,65 No. 6, No. 9,383,353, No. 9,228,225, No. 9,034,920, No. 8,753,814, No. 8,163,910, No. 8,030,096 No. 7,767,829, No. 7,704,284, No. 7,563,907, No. 7,402,671, No. 7,256,291, No. 7,038,063 No. 6,750,346, No. 6,743,568, No. 6,399,392, No. 6,225,050, No. 6,191,278, No. 6,048,982 The disclosures are described in U.S. Patent Publications No. 5,286,803 and No. 4,945,171; and in one or more of U.S. Patent Application Publications No. 2020 / 0277670, 2011 / 010575, 2006 / 0199949, and 2003 / 0165942, the disclosures of which are incorporated herein by reference in their entirety.

[0091] If desired, the donor and / or acceptor fluorophores present in the dye structure of the embodiments of the present invention may be substituted with one or more water-soluble groups (WSGs), for example, as defined above.

[0092] The dye structures found to be used in embodiments of the present invention are further described in U.S. Patent Nos. 11,702,547, 11,214,688, and 10,844,228; and published U.S. Patent Applications Nos. 2022 / 0348770 and 2023 / 0152327; and pending U.S. Patent Applications Nos. 18 / 120,554, 18 / 127,568, and 18 / 201,940; the disclosures thereof are incorporated herein by reference.

[0093] Quencher As described above, the nucleic acid probe may include a quencher 106 in an energy transfer relationship with one or more acceptor fluorophores 112. The quencher 106 may be ligated to the terminal (e.g., 5' or 3' end) of the nucleic acid 102. In certain embodiments, the quencher 106 is ligated to the 3' end of the nucleic acid 102. Certain embodiments include an internal quencher 106 ligated to a nucleotide that is not located terminally. The internal quencher may be located 7 to 15 nucleotides away from the dye structure (e.g., 7 to 15 nucleotides from the 5' end of the nucleic acid). In some specific embodiments, the internal quencher is located 9 or 10 nucleotides from the 5' end of the nucleic acid. The internal quencher can be used together with or in place of a terminal quencher (e.g., a quencher at the 5' or 3' end).

[0094] The quencher may include any convenient absorbent moiety. The quencher may be a small molecule quencher. In certain cases, the quencher is a fluorescent dye having an emission distribution significantly longer than the emission maxima of the donor fluorophore (e.g., the acceptor and / or donor fluorophore described above). In certain embodiments, the quencher is a non-fluorescent moiety. In certain embodiments, the absorption and / or excitation distribution of the quencher includes the emission maxima of the acceptor fluorophore described above (e.g., about 500 nm to 875 nm). The absorption and / or excitation distribution of the quencher may further include the emission maxima of the donor fluorophore described above (e.g., about 450 nm to 725 nm). Therefore, the quencher can absorb light in the range of approximately 450 nm to approximately 875 nm, for example, 440 nm to 485 nm, 485 nm to 510 nm, 510 nm to 565 nm, 565 nm to 590 nm, 590 nm to 625 nm, 625 nm to 740 nm, or 740 nm to 875 nm.

[0095] The quencher can be selected from Black Hole Quencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher. In certain embodiments, the quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphoramidite quencher, azoquencher, anthraquinone quencher, their conjugates, and combinations thereof.

[0096] The specific quencher used can be selected based on the desired parameters, such as (but not limited to) excitation maxima, emission wavelength maxima, Stokes shift, extinction coefficient, and / or any other relevant factors.

[0097] Such quenchers are described in one or more of U.S. Patents 11,242,554, 10,886,006, 9,540,515, 8,916,345, 8,410,255, 7,956,169, 7,879,986, 7,803,536, 7,439,341, 7,166,715, 7,019,129, 6,790,945, 6,699,975, 6,399,392 and 6,323,337, the disclosures of which are incorporated herein by reference in their entirety.

[0098] Further aspects of the probe Certain embodiments of the probe may include features that increase binding or hybridization effectiveness with a target sequence. Such modifications may include minor groove binders (MGBs). Certain embodiments may include one or more, two or more, or any other number of MGBs on the probe. The MGBs may be ligated to the 5' or 3' end of the nucleic acid. For the purposes of this disclosure, 10 -3 M -1If a molecule can bind to the secondary groove of double-stranded DNA with the above association constants, it is considered an MGB. This type of binding can also be detected by established spectrophotometric methods such as ultraviolet spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, as well as by gel electrophoresis.

[0099] MGB may include certain naturally occurring compounds such as nettropsin, dystamycin, lexitropsin, mitramycin, chromomycin A3, olibomycin, anthramycin, and cibilomycin, as well as further related antibiotics and synthetic derivatives. It may also include certain bisquaternary ammonium heterocyclic compounds, diaryl amidines such as pentamidine, stilvamidine, and berenyl, CC-1065, and related pyrroloindole and indole polypeptides, Hoechst33258, and 4'-6-diamidino-2-phenylindole (DAPI). Various oligopeptides consisting of naturally occurring or synthetic amino acids are subgroove binder compounds including 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI) and N-methylpyrrole-4-carboxy-2-amide (MPC). Certain embodiments include dimers, trimers, tetramers, and pentamers of CDPI. Further embodiments include dimers, trimers, tetramers, and pentamers of MPC. Further examples of MGB can be found in U.S. Patents 5,801,155 and 7,582,739, the disclosures of which are incorporated herein by reference in their entirety.

[0100] Probes for nucleic acid detection can be assembled as a panel of probes for multiple detection. In such cases, the nucleic acids of each probe are complementary to different target sequences; for example, the first nucleic acid is complementary to the first target sequence, and the second nucleic acid is complementary to the second target sequence, where the first target sequence is different from the second target sequence. The first and second target sequences may be located on the same molecule (e.g., a chromosome) or on different molecules. In certain cases, the first and second target sequences are located within the same amplicon (e.g., a region amplified by a set of primers). Furthermore, different combinations of donor and / or acceptor fluorophores can be used for each probe to distinguish between probes in the panel. Certain embodiments use the same donor fluorophore but acceptor fluorophores with different emission wavelengths. Such a situation allows for simultaneous detection of probes with only one excitation source. Other options for simultaneous detection may include donor fluorophores having similar excitation and emission distributions. Certain embodiments have probes that use donor and / or acceptor fluorophores having different excitation or emission distributions. In such situations, excitation or detection may occur asynchronously. Those skilled in the art will understand which donor and / or acceptor fluorophores, as well as how to select appropriate excitation and detection.

[0101] A panel of probes may contain as many probes as possible that can be identified by simultaneous and / or asynchronous detection. For example, a finite number of fluorophores may have the same excitation maxima but different emission maxima, thus limiting the amount of simultaneous detection. Thus, a particular panel may use multiple sets of such fluorophores such that a first excitation source allows detection of some probes, and a second excitation source allows detection of several more probes. Examples of such fluorophores can be found in U.S. Patent Applications 18 / 201,940 and 18 / 120,554, the disclosures thereof which are incorporated herein by reference in their entirety.

[0102] The number of distinct probes in a given panel can vary. In some cases, the number may be 2 or more, e.g., 5 or more, e.g., 10 or more, e.g., 25 or more, 50 or more, 75 or more, etc., and in some cases, the number may be 1000 or less, e.g., 500 or less, e.g., 250 or less, e.g., 200 or less, 150 or less, e.g., 100 or less. The number of probes in a panel may be 5-10 probes, 5-15 probes, 5-25 probes, 10-20 probes, 10-25 probes, 10-30 probes, 10-35 probes, 10-40 probes, 10-45 probes, 10-50 probes, 15-25 probes, 15-35 probes, 15-45 probes, and / or any other range of probes. A panel of probes can target a specific set of genes so that the panel has diagnostic or prognostic capabilities for diseases such as cancer. Further panels can focus on the Recommended Uniform Screening Panel (RUSP), a list of disorders recommended by the Director General of the Department of Health and Human Services as part of a universal newborn screening program. Specific panels can target traces of microorganisms in a sample to diagnose, for example, infectious agents and, selectively, their variants. For instance, a panel could be used to identify tuberculosis and some of its antibiotic-resistant variants.

[0103] A panel of probes can be added to a single reaction, such as a real-time or quantitative PCR reaction. In certain cases, the panel is provided as an assembly of individual probes, with each probe individually packaged or separated; in other cases, the panel is provided as a pooled assembly of all probes within that panel. In either the pooled or individually packaged cases, the panel may include components to aid stability and / or detection. Such components may include one or more of the following to avoid photobleaching: buffers, primers, polymerases, nucleotides, thickeners, and fluorescence stabilizers. With or without additional components, the panel can be provided in a dry state (e.g., lyophilized) or in a suitable medium. The suitable medium may be aqueous (e.g., water-based) or organic. In the pooled embodiment, the probes may be optimized or modified.

[0104] As discussed above, the adjustability of the embodiment can support multiple applications. Such adjustability can compensate for detection problems, such as a decrease in emission and / or signal brightness from specific fluorophores, and for variations within a sample, such as the simultaneous detection of small and large amounts of targets within a sample. Thus, probes in a panel of probes may have a dye structure with varying numbers of donor and / or acceptor fluorophores to compensate for variations in brightness and / or emission from probes in the panel.

[0105] The dye structure of a particular embodiment may differ. In some cases, the dye structure of a particular embodiment includes a polymer backbone; one or more pendant donor fluorophores, each independently linked to a repeating unit of the polymer backbone; and one or more pendant acceptor fluorophores, also linked to the repeating unit of the polymer backbone, that are in an energy transfer relationship with the pendant donor fluorophores. Thus, the dye structure of some embodiments includes one or more pendant donor fluorophores and one or more pendant acceptor fluorophores, both linked to a common polymer backbone, configured to be in energy-accepting proximity to one or more pendant donor fluorophores. In some embodiments, there may be multiple pendant donor fluorophores, configured to be in energy-transferring proximity to the pendant acceptor fluorophores, and in some cases, the number of pendant donor fluorophores may range from 2 to 20, for example, 2 to 15, for example, 2 to 10. The term "pendant group" refers to a side-chain group that is linked to the backbone but is not part of the backbone itself. In embodiments of the dye structure, the donor fluorophores can transfer energy to the linked acceptor fluorophores. Therefore, the target dye structure includes a linked acceptor signaling fluorophore that is in energy-receptor proximity to the donor fluorophore system, i.e., in energy-receptor proximity to at least one linked donor fluorophore. The specific configuration of the pendant group can be determined and controlled by the arrangement of repeating units of the underlying polymer backbone (also called the "modular scaffold") to which the pendant group is bound. The dye structure may include multiple water-solubilizing groups bound to the scaffold and / or pendant group at any convenient positions to provide a water-soluble polymer dye. The polymer backbone, i.e., the modular scaffold, can consist of repeating units that form a polymer backbone having side-chain groups to which the pendant group can be bound. The repeating units can be arranged in various configurations to provide a dye structure with desired spectral properties.The distance and arrangement between the covalent bonding sites of the pendant donor fluorophore and the acceptor fluorophore (if any) can be controlled to provide a desired energy transfer process.

[0106] As described above, the polymer backbone and / or pendant fluorophores (i.e., donor and acceptor fluorophores) may contain one or more water-solubilizing groups (WSGs), if desired. In some cases, the WSG is a pendant group directly attached to the polymer backbone, for example, as a side chain of the polymer backbone. In specific cases, the WSG is a substituent bonded to the pendant donor fluorophore or the pendant acceptor fluorophore. In some cases, each of the pendant donor fluorophore groups is substituted with one or more WSGs. As used herein, the terms “water-solubilizing group,” “water-soluble group,” and “WSG” are used interchangeably and refer to a group or substituent that is well solvable in an aqueous environment, e.g., under physiological conditions, and confers improved water solubility to the molecule to which it is bound. WSGs can increase the solubility of a given dye structure, primarily in aqueous solutions, compared to a control dye lacking WSGs. The water-solubilizing group can be any convenient hydrophilic group that is well solvable in an aqueous environment. The water-soluble dye structures of this disclosure have solubility under aqueous conditions that makes them particularly suitable for application to a variety of biological assays. Various water-soluble polymer groups can be adapted for use in the WSG of the target dye. By including any suitable water-soluble group (WSG) in the dyes described herein, increased water solubility can be provided, for example, as described above.

[0107] As summarized above, the dye structures of embodiments of the present invention include a plurality of pendant donor fluorophores and one or more pendant acceptor fluorophores. In some cases, the number of donor fluorophores exceeds the number of acceptor fluorophores. In certain embodiments of the dye structure of interest, the ratio of donor fluorophores to acceptor fluorophores is selected from 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 20:2. In a particular case, the ratio of donor fluorophores to acceptor fluorophores is 5:1. In a particular case, the ratio of donor fluorophores to acceptor fluorophores is 6:1. In a particular case, the ratio of donor fluorophores to acceptor fluorophores is 7:1. In a particular case, the ratio of donor fluorophores to acceptor fluorophores is 8:1. In a particular case, the ratio of donor fluorophores to acceptor fluorophores is 9:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 10:1.

[0108] As described above, in the dye structure of the present invention, the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship. Therefore, in embodiments of the present invention, excitation of the donor can result in energy transfer to the covalently bonded acceptor signaling fluorophore and emission therefrom. Mechanisms for energy transfer between the donor chromophore and the linked acceptor signaling fluorophore include, for example, resonance energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET) and quantum charge exchange (Dexter energy transfer). These energy transfer mechanisms can be in a relatively short range, i.e., the proximity of the chromophores in a light-gathering multichromophore system to each other and / or to the acceptor fluorophore enables efficient energy transfer. Under conditions for efficient energy transfer, amplification of emission from an acceptor fluorophore can occur, where the emission from the emitting acceptor fluorophore is stronger when the incident light ("pump light") is at a wavelength absorbed and transferred from the chromophore of a condensing chromophore than when the emitting acceptor fluorophore is directly excited by the pump light. "Efficient" energy transfer means that 10% or more of the energy collected by the donor chromophore is transferred to the acceptor, e.g., 20% or more, or 30%, 40%, or 50% or more. "Amplified" means that the signal from the acceptor fluorophore is 1.5 times or more when excited by energy transfer from the donor condensing chromophore system compared to direct excitation of the acceptor fluorophore with incident light of equivalent intensity. The signal can be measured using any convenient method. In some cases, a signal of 1.5 times or more refers to the intensity of the emitted light. In specific cases, a signal of 1.5 times or more refers to an increase in the signal-to-noise ratio.In certain embodiments of the dye structure, the acceptor fluorophore emission, when excited by the chromophore, is 1.5 times or more, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 20 times, 50 times, 100 times or more, compared to the direct excitation of the acceptor fluorophore by incident light.

[0109] The dye structures of embodiments of the present invention may have any convenient molecular weight (MW). In some cases, the MW of the dye structure may be expressed as the average molecular weight. In some cases, the dye structures have an average molecular weight in the range of 500 to 500,000, e.g., 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000, or even an average molecular weight in the range of 50,000 to 100,000 Daltons. In some cases, the polymer fluorescent dyes have molecular weights in the range of 5 to 75 kDa, 10 to 50 kDa, e.g., 15 to 45 kDa.

[0110] In the embodiment, the target dye structure provides fluorescence emission from acceptor fluorophores that is brighter than the emission possible from such a fluorescent dye alone. The emission of the dye structure is 50 mM -1 cm -1 For example, 60 mM -1 cm -1 Above 70mM -1 cm -1 Above 80mM -1 cm -1 Above 90mM -1 cm -1 Above 100mM -1 cm -1 More than 150mM -1 cm -1 Above 200mM -1 cm -1 more than 250mM -1 cm -1 Above 300mM -1 cm -1The luminance can be greater than or equal to that of the dye structure. In certain cases, the luminescence of the dye structure may be at least five times greater than the luminance of a directly excited acceptor fluorophore, for example, at least ten times, at least twenty times, at least thirty times, at least fifty times, at least 100 times, at least 300 times, or even greater than the luminance of a directly excited acceptor fluorophore.

[0111] In addition to the attributes described above, the dye structures of the embodiments of the present invention may have one or more additional desired spectral properties, such as a specific emission maximum wavelength, extinction coefficient, and quantum yield.

[0112] The diverse emission profiles depend on various factors such as the selected comonomers, linking groups, substituents, and linking acceptor fluorophores that constitute the dye structure.

[0113] In some embodiments, the dye structure may include a segment having a formula described in PCT / US2019 / 024662 (published as International Publication No. 2019 / 191482) and one or more U.S. Patent applications No. 18 / 201,940 and No. 18 / 120,554, the disclosures of which are incorporated herein by reference in their entirety. Further details regarding the WSG group that may be found in the dye structures of embodiments of the present invention can be found in PCT application PCT / US2019 / 024662, published as International Publication No. 2019 / 191482.

[0114] The general structure of the obtained nucleic acid probe is shown by the following formula: [ka] nucleic acid (In the formula, Each D is independently a pendant donor fluorophore (e.g., as described herein); Each A is independently an acceptor chromophore (e.g., as described herein); l, m, and n are each independent integers between 1 and 20; L is the linker; R1 and R2 are independently selected from terminal groups, polymer segments, donor chromophore groups, water-solubilizing groups, linkers, and linkers for specific binding members; The nucleic acid is a nucleic acid having a sequence complementary to the target nucleic acid (for example, those described herein); and Q is a quencher (for example, as described herein).

[0115] In some cases of the aforementioned formulas, l, m, and n are independently between 1 and 10, for example, 2 to 20, 3 to 10, or 3 to 6. In some cases of the aforementioned formulas, n is an integer between 2 and 20, for example, 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In certain embodiments of the aforementioned formulas, m is 1.

[0116] Figure 2 shows one exemplary (and non-limiting) depiction of a nucleic acid probe according to a particular embodiment. The example in Figure 2 provides a nucleic acid having a dye structure bound to a first end of the nucleic acid and a quencher bound to a second end of the nucleic acid. The dye structure includes a non-conjugated polymer backbone formed of amino acids, e.g., a peptide structure. This example provides six donor fluorophores and one acceptor fluorophore, as described above.

[0117] method Aspects of the present invention include a method for evaluating a sample for the presence of a target nucleic acid. Aspects of the method include preparing a reaction mixture comprising a sample and a nucleic acid probe. Such probes are described in the preceding disclosure and can be used to prepare the reaction mixture. In certain cases, the sample contains, is suspected of containing, may contain, or does not contain a nucleic acid. In certain circumstances, the sample contains one or more copies of a target nucleic acid for which the nucleic acid probe is complementary. In certain embodiments, the nucleic acid probe hybridizes to any copy of the target nucleic acid in the sample. Further embodiments monitor the reaction mixture for a signal emitted from a dye structure associated with the nucleic acid probe in order to assay the sample for the presence of a target nucleic acid.

[0118] In some cases, this method may involve irradiating the reaction mixture at the excitation wavelength of one or more donor fluorophores. Specific wavelengths, bandwidths, intensities, or other parameters may be selected by those skilled in the art and may depend on which donor fluorophores are part of the nucleic acid probe.

[0119] In some cases, the reaction mixture further comprises a polymerase having 5'-3' nuclease activity, and monitoring the reaction mixture involves performing a nucleic acid amplification reaction. Nucleic acid amplification may include isothermal amplification and polymerase chain reaction (PCR). In the amplification reactions described herein, the polymerase having 5'-3' nuclease activity polymerizes a new chain based on a target sequence or target region. When the polymerase encounters the hybridized nucleic acid probe, the nuclease activity hydrolyzes the nucleic acid that is part of the nucleic acid probe. This hydrolysis releases a dye structure from the quencher, causing the dye structure to release a signal.

[0120] As is known to those skilled in the art, PCR comprises providing a pair of amplification primers and deoxynucleotide triphosphates to a reaction mixture, wherein the first amplification primer contains a sequence complementary to a region of one strand of the target nucleic acid and can prime the synthesis of the complementary strand, and the second primer contains a sequence complementary to a region of the second strand of the target nucleic acid and can prime the synthesis of the complementary strand. In such embodiments, a polymerase exhibiting 5'-3' nuclease activity amplifies the target nucleic acid between the amplification primers, and the nucleic acid probe is complementary to a region of the target nucleic acid located between the amplification primer pair. PCR may further comprise cycling the reaction temperature of the sample to a first temperature suitable for denaturing the target nucleic acid, then a second temperature suitable for allowing the hybridization of the amplification primer pair and the nucleic acid probe into the target nucleic acid, and then a third temperature suitable for the polymerase to amplify the target nucleic acid. The cycling step can be repeated for any number of iterations sufficient to allow the signal to be released from the dye structure. In certain embodiments, the reaction temperature cycle is repeated over 4 to 35 iterations, for example, at least 4 iterations, at least 5 iterations, at least 10 iterations, at least 15 iterations, at least 20 iterations, or at least 25 iterations.

[0121] The reaction temperature for PCR can be selected to suit the specific type of reaction, the reaction mixture, and / or the components contained within the reaction mixture. In PCR, the first temperature for denaturation may be above the temperature for separating any double-stranded nucleic acid. This first temperature may be 100°C or close to it, for example, 90°C, 92°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C. However, in certain situations that can be understood by those skilled in the art, lower temperatures may be used. A second temperature suitable for enabling primer hybridization or annealing is typically the melting temperature (T) of the primer and / or probe. mThe temperature is below 55°C. Typically, this range is between 55°C and 72°C, although certain embodiments may use lower temperatures. Furthermore, this second temperature can be adjusted between iterations in step-up PCR (increasing the temperature with each iteration), touchdown PCR (decreasing the temperature with each iteration), and / or any other cycling strategies to improve sensitivity and / or detection. Finally, the third temperature suitable for extension depends on the specific enzyme to ensure processability and / or efficiency in the extension or polymerization of the new nucleic acid chain. This temperature is typically around 72°C, but can be varied depending on the enzyme. Thus, the third temperature can be selected from about 65°C to about 80°C, for example, 70°C, 72°C, 75°C, and / or 78°C.

[0122] Certain cases involve implementing probes in real-time PCR, including quantitative PCR (qPCR), as described herein. While real-time PCR and qPCR can use nonspecific dyes to quantify amplification, these methodologies may measure off-target amplification and therefore have limited accuracy. The probes described herein improve qPCR accuracy by detecting specific amplicons. Such probes hybridize or anneal to a target sequence (i.e., the complementary sequence of the probe) during the same step as primer hybridization. During the extension step, a polymerase having 5'-3' exonuclease activity can hydrolyze or digest the nucleic acid portion of the probe. Hydrolysis releases a scaffolded chromophore or dye structure from the probe, allowing the dye structure to diffuse within the reaction mixture. The release disrupts the energy transfer relationship between the acceptor fluorophore and the quencher on the dye structure, thus enabling the measurement or imaging of the fluorescence signal. In qPCR reactions, imaging or detection can be performed continuously throughout the reaction or at a single point in time during a temperature cycle. For example, imaging can be performed after extension but before denaturation. However, imaging or detection can be performed during denaturation and / or hybridization. Further details regarding real-time PCR and / or qPCR as found to be used in embodiments of the present invention are described in U.S. Patents 11,001,877, 9,944,978, 8,697,362, 8,137,616, 8,017,380 and 7,101,663; and further in published U.S. Patent Applications Publications 2020 / 0370097, 2012 / 0088691, 2011 / 0300544 and 2005 / 0053950, the disclosures of which are incorporated herein by reference in their entirety.

[0123] Isothermal amplification can be carried out via various methods known in the art, such as nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand substitution amplification (SDA), recombinase polymerase amplification (RPA), and rolling circle amplification (RCA). In addition to polymerases having 5'-3' nuclease activity, such reaction mixtures may include one or more primers, ligases, recombinases, deoxyribonucleotides, ribonucleotides, one or more adapters, and various other components known to those skilled in the art. The isothermal amplification reaction temperature may depend on the type of reaction, the enzyme, and / or any other factors. In certain embodiments, the reaction is carried out at temperatures between about 30°C and 45°C, for example, 32°C, 35°C, 37°C, 40°C, and 42°C. Certain embodiments use 37°C as the reaction temperature for isothermal amplification reactions.

[0124] Nucleic acid amplification, such as that described herein, may further include reaction-adjunct components, including buffers, solvents, and / or any other components. Such buffers may include HEPES, PBS, SSC, Tris, any other buffers for specific enzymes, and combinations thereof. Solvents may also be used to enhance specificity, reaction efficiency, and / or any other properties that aid such reactions. A common solvent used in many reactions is dimethyl sulfoxide (DMSO). As those skilled in the art will understand, additional components may be part of the reaction mixture.

[0125] Furthermore, the method described herein can be used in multiplexing reactions with a second nucleic acid probe described herein. In the second probe, the relevant nucleic acid is complementary to a second target sequence that is different from the first target sequence. When used in amplification reactions, the second probe can hybridize to a region between the amplification primer pairs of the first nucleic acid probe or between the amplification primer pairs of the second probe. In certain multiplexing embodiments, the number of different target nucleic acids assayed simultaneously varies, in some cases being two or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, 50 or more, 75 or more, etc., and in some cases being 1000 or less, e.g., 500 or less, e.g., 250 or less, e.g., 200 or less, e.g., 150 or less, e.g., 100 or less. The number of target nucleic acids to be assayed may be 5–10 targets, 5–15 targets, 5–25 targets, 10–20 targets, 10–25 targets, 10–30 targets, 10–35 targets, 10–40 targets, 10–45 targets, 10–50 targets, 15–25 targets, 15–35 targets, 15–45 targets, and / or any other range of targets.

[0126] In some embodiments, a sample is any one that contains or may contain nucleic acids. Such samples can be biological or environmental. Biological samples include animals, plants, fungi, bacteria, viruses and / or archaea. A sample can be an entire organism, a single cell, or another part thereof. A particular sample may originate from a single tissue, an organ, a biological product (e.g., blood), or any combination thereof. In certain embodiments, a biological sample is collected as saliva, feces, urine, sweat, sebum, mucus, semen, milk, blood, and / or any other biological product. Blood samples can be collected as peripheral whole blood, peripheral whole blood with red blood cells lysed before cell isolation, umbilical cord blood, bone marrow, or density gradient purified peripheral blood mononuclear cells. Certain biological samples may include cell and / or tissue cultures such as CD34+ cells, HEK cells, HeLa cells, and / or any other cell lines. Tissue samples may include normal tissue (e.g., liver, kidney, brain, skin, breast, fat, bone, bone marrow, etc.) or abnormal tissue (e.g., neoplasms, cancer, tumors, diseased tissue, etc.).

[0127] Environmental samples can be used to identify the presence of a particular species or organism without direct visual observation or other evidence of its presence. Such samples may include those collected from air, water, sewers, ground, toilets, sinks, doorknobs, and / or any other non-biological locations or materials.

[0128] Fluorescence in a sample can be measured using a fluorophotometer. In some cases, excitation radiation from an excitation source having a first wavelength passes through an excitation optical system. The excitation optical system generates excitation radiation that excites fluorophores in the reaction mixture. In response, a fluorescently labeled target in the sample emits radiation having a different wavelength from the excitation wavelength. The collection optical system then collects the emission from the reaction mixture. The apparatus may include a temperature controller to maintain the sample at a specific temperature while the sample is being scanned. In certain cases, a multi-axis translational stage moves a microtiter plate holding multiple samples to position different wells to be exposed. The multi-axis translational stage, temperature controller, autofocus function, and electronics related to imaging and data acquisition can be managed by a properly programmed digital computer. The computer can also convert the data collected during the assay into another format for presentation.

[0129] In some embodiments, a method for evaluating a sample for the presence of a target nucleic acid further includes detecting fluorescence in a flow cytometer. In some embodiments, a method for evaluating a sample for the presence of a target nucleic acid further includes imaging a sample in contact with a labeled composition using a fluorescence microscope. Fluorescence microscopy imaging can be used to identify polymer dye conjugate-target nucleic acid binding complexes in the contacted sample to assess the presence of the target analyte. Microscopy techniques intended for use in the methods in question include laser scanning confocal microscopy.

[0130] Since specific methods for detecting target nucleic acids utilize nucleic acid amplification, fluorescence can be detected via a real-time thermal cycler or a thermal cycler equipped with excitation and detection arrays to monitor fluorescence emission from the reaction mixture.

[0131] Hybridization products Certain embodiments of this disclosure include products prepared using nucleic acid probes disclosed herein. Such products may include probes hybridized to a target nucleic acid. Such products may include a first nucleic acid and a second nucleic acid, each containing a plurality of nucleotides forming a chain from the 5' end to the 3' end. Furthermore, the first nucleic acid is complementary to at least a portion of the second nucleic acid. The first nucleic acid may be the nucleic acid probe described herein and include the dye structure and quencher described herein. The first nucleic acid may include the second quencher, a secondary groove binder, a water-soluble group, etc., as described above.

[0132] The second nucleic acid may include a target nucleic acid. Such nucleic acid may be DNA, RNA, or another form of nucleic acid (e.g., those described herein). Such targets may be nuclear and / or genomic DNA, messenger RNA (mRNA), pre-mRNA, ribosomal RNA, transfer RNA, amplicons, and / or any other nucleic acid that can be targeted and / or is of interest.

[0133] system Aspects of the present invention further include systems for use in carrying out the methods and compositions of the present invention. A sample analysis system may include a chamber for holding the sample described herein. A system of such an embodiment may include an illumination source directed to strike the sample, and a detector directed so that the light emitted from the sample strikes the detector. The sample may include a nucleic acid probe as described, and may contain or be suspected of containing a target nucleic acid. Further embodiments of the system may include a heating element for altering the temperature of the sample. Such a heating element may include a water bath or a heating block, and may alter the temperature of the sample using a thermoelectric effect.

[0134] Further embodiments include a user interface that allows selection of temperature, illumination wavelength, detection wavelength, and temperature cycle program. Such a user interface may be analog, digital, and / or graphical. Examples of such systems may include thermal cyclers, plate readers, flow cytometers, fluorescence microscopes, and / or any other systems that meet the requirements of this specification.

[0135] In certain embodiments, the system may further include a computer-based system configured to detect the presence of a fluorescent signal. “Computer-based system” means hardware means, software means, and data storage means used to analyze the information of the present invention. The minimum hardware of the computer-based system of the present invention includes a central processing unit (CPU), input means, output means, and data storage means. Those skilled in the art will readily understand that any of the currently available computer-based systems is suitable for use in the system in question. The data storage means may include any product containing the recording of the information described above, or memory access means capable of accessing such product.

[0136] "Recording" data, programming, or other information on a computer-readable medium refers to the process of storing information using any such method known in the art. Any convenient data storage structure can be selected based on the means used to access the stored information. For example, various data processor programs and formats such as word processing text files and database formats can be used for storage.

[0137] "Processor" refers to any combination of hardware and / or software that performs the necessary functions. For example, any processor as used herein may be a programmable digital microprocessor, such as one available in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). If the processor is programmable, appropriate programming can be communicated to the processor remotely or stored in advance on a computer program product (e.g., a portable or fixed computer-readable storage medium, whether magnetic, optical, or solid-state based). For example, a magnetic medium or optical disc can carry programming that can be read by a suitable reader communicating with each processor at its corresponding station.

[0138] For example, in addition to the sensor device and signal processing module described above, the system of the present invention may include several additional components such as data output devices such as monitors and / or speakers, data input devices such as interface ports and keyboards, fluid processing components, and power supplies.

[0139] Other systems may find use in carrying out the method in question. In certain embodiments, the system may be a fluorometer or microscope loaded with a sample having a fluorescent composition of any of the embodiments discussed herein. The fluorometer or microscope may include a light source configured to direct light onto the assay area of ​​the channel or sample field. The fluorometer or microscope may also include a detector configured to receive a signal provided by the fluorescent composition from the assay area of ​​the channel or field.

[0140] kit Aspects of the present invention further include kits for use in carrying out the methods and compositions of the present invention. Compositions of the present invention, for example, one or more probes (such as those described above), may be included as reagents in the kit as starting materials, or may be provided for use in the methodology described above, for example. The kit may include, for example, probes and containers as described above. Any convenient container may be used, such as tubes, bottles, or wells in multiwell strips or plates, boxes, bags, or insulated containers. The kit of the present invention may further include one or more components selected from probes for a given target sequence, support-bound probes, cells, supports, biocompatible aqueous elution buffers, controls (positive and / or negative), and, optionally, instructions for use. A given kit may include reagents suitable for the detection of a single target nucleic acid, or multiple reagents suitable for the detection of two or more different target nucleic acids, for example, if the given kit is configured for multiple detection applications.

[0141] In certain embodiments, the kit is used to evaluate a sample for the position of a target sequence, such as in situ hybridization. Therefore, in some cases, the kit includes one or more components suitable for cell permeabilization or lysis. One or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in multiwell strips or plates).

[0142] In certain embodiments, the kit further comprises reagents for carrying out nucleic acid amplification. The reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting the sample with the nucleic acid probe, wash buffers, control reagents, enzymes (e.g., polymerase, polymerase having 5'-3' nuclease activity, recombinase, ligase, etc.), nuclease-free water, nucleotide triphosphates, deoxynucleotide triphosphates, one or more primers, adapters, and combinations thereof. Furthermore, the kit may include cell-permeable reagents such as methanol, acetone, or surfactants (e.g., Triton, NP-40, saponin, Tween20, digitonin, leucoperme, or any combination thereof or buffers).

[0143] The kit's compositions may be provided in liquid compositions, such as any suitable buffer. Alternatively, the kit's compositions may be provided in dry compositions (e.g., lyophilized), and the kit may optionally include one or more buffers for reconstituting the dry compositions. In certain embodiments, the kit may include aliquots of the compositions provided in separate containers (e.g., separate tubes, bottles, or wells in multiwell strips or plates).

[0144] Furthermore, one or more components may be combined in a single container, such as a glass or plastic vial, tube, or bottle. In certain cases, the kit may further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) in which all components (and their separate containers) reside. The kit may further include packaging, either separate from or attached to the kit container, on which information about the kit, the components of the kit, and / or instructions for using the kit are printed.

[0145] In addition to the components described above, the kit may further include instructions for carrying out the method. These instructions may be present in the kit in various forms, and one or more of these forms may be present in the kit. One possible form of these instructions is printed information on a suitable medium or substrate, e.g., one or more sheets of paper on which the information is printed, the kit packaging, or an accompanying document. Yet another means may be a computer-readable medium on which the information is recorded, e.g., a diskette, CD, DVD, or portable flash drive. Yet another possible means may be a website address that can be used via the internet to access information located remotely. Any convenient means may be present in the kit.

[0146] usefulness The scaffolded chromophores for nucleic acid detection and nucleic acid probes, compositions, methods, and systems described herein can be found in a variety of applications, including diagnostic and research applications, where labeling, detection, and / or analysis of a target of interest is desired. Such applications include methodologies such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), evaluation of free analytes, and evaluation of receptor-binding ligands. The compositions, systems, and methods described herein, but not limited to, may be useful for the analysis of a number of samples, including biological fluids, cell culture samples, and tissue samples. In certain embodiments, the compositions, systems, and methods described herein can be found in methods for detecting the presence of an analyte in a sample using fluorescent labeling, such as in fluorescence-activated cell sorting or analysis, immunoassays, and immunostaining. In specific cases, the compositions and methods are found in applications where the purpose is to evaluate a sample for the presence of a target analyte.

[0147] In some cases, the method and composition may be used in any assay format where the purpose is the detection and / or analysis of a target from a sample, including but not limited to quantitative PCR, flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography. In specific cases, the method and composition may be used in any application where the purpose is the fluorescent labeling of a target molecule. The composition may be adapted for use in any convenient application where a nucleic acid probe is found.

[0148] The following examples are provided as illustrations, not as limitations. [Examples]

[0149] Example 1 - Real-time PCR Various nucleic acid probes are expected to be incorporated into real-time PCR (rtPCR). rtPCR can be quantitative and may be referred to as quantitative PCR (qPCR). In rtPCR, a reaction mixture is prepared according to a standard PCR reaction, with the addition of probes that can identify specific sequences that may be present in the sample. The reaction mixture contains a sufficient amount of buffer, nucleotides (e.g., deoxyribonucleotides and / or ribonucleotides), polymerase (e.g., taq polymerase), primers, templates, and probes, such as those shown in Figure 2. The reaction mixture may contain the following components and amounts: 10 μL of template or sample Forward primer (5 μM) 0.5 μL Reverse primer (5 μM) 0.5 μL dNTP (10mM) 0.5μL 10X buffer 2.5μL Taq polymerase (exo+) 0.5 μL Probe (Figure 2) (2.5 μM) 0.5 μL 10μL of water Total 25μL

[0150] Once the reaction mixture is prepared, it is incubated in a suitable thermal cycler, which typically has a light source and detector that allow for the excitation of fluorophores and the detection of any fluorophore emission. The thermal cycler is cycled through temperatures that allow for denaturation, annealing of primers and probes, and primer extension. The cycled reaction can follow the following program: (1) Initial denaturation: 3 minutes at 95°C (2) Denaturation: 95°C for 1 minute (3) Annealing at 55°C for 2 minutes (4) Extension: 3 minutes at 72°C (5) Imaging / Detection (6) Repeat cycle steps 2-5 for 10-45 cycles.

[0151] During extension, the exonuclease activity of polymerase hydrolyzes the nucleic acid, thus releasing the donor and acceptor fluorophores from the nucleic acid. This release of the fluorophores allows them to diffuse from the quencher. This diffusion eliminates the energy transfer relationship between the fluorophores and the quencher, enabling luminescence.

[0152] The reaction is monitored at appropriate excitation and emission maxima. Using the probe shown in Figure 3, the appropriate excitation wavelengths include the excitation maxima of the six donor fluorophores. The detection wavelengths include the emission maxima of the acceptor fluorophores. Monitoring can be performed consistently through the reaction program or during separate detection or imaging steps.

[0153] Notwithstanding the attached claims, this disclosure is also defined by the following clauses: Clause 1. A probe for nucleic acid detection, A nucleic acid complementary to the target sequence, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A dye structure linked to the first end of the nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. Pigment structures including; and A quencher ligated to the second end of the nucleic acid, which is in an energy transfer relationship with one or more acceptor fluorophores; A probe for nucleic acid detection, including [specific component]. Clause 2. The probe described in Clause 1, wherein the nucleic acid comprises one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA). Clause 3. The probe according to Clause 1 or 2, wherein the base associated with each nucleotide in the plurality of nucleotides is selected from canonical bases, modified bases, and base analogs. Clause 4. The probe according to Clause 3, wherein at least one nucleotide among the plurality of nucleotides is a nucleotide analog. Clause 5. The probe according to any one of Clauses 1 to 4, further comprising a sub-groove binder (MGB) linked to the nucleic acid. Clause 6. The probe according to Clause 5, wherein MGB is ligated to the 3' end of the nucleic acid. Clause 7. A probe as described in any of the preceding clauses, wherein the plurality of nucleotides comprises 10 to 50 nucleotides. Clause 8. The probe according to Clause 7, wherein the plurality of nucleotides comprises 18 to 30 nucleotides. Clause 9. The probe according to any of the preceding clauses, wherein one or more excitable donor fluorophores have an excitation maximum in the range of 300 to 700 nm. Clause 10. The probe according to Clause 9, wherein one or more excitable donor fluorophores have excitation maxima in the range of 300-380 nm, 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-700 nm. Clause 11. The probe according to Clause 9 or 10, wherein one or more excitable donor fluorophores have emission maxima in the range of 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-725 nm. Clause 12. The probe according to any of the preceding clauses, wherein the one or more excitable donor fluorophores are selected from the group consisting of bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores. Clause 13. The probe according to any of the preceding clauses, wherein one or more excitable donor fluorophores are bonded to a water-soluble group. Clause 14. The probe according to Clause 13, wherein the water-soluble group comprises a nonionic water-soluble group. Clause 15. The probe according to Clause 14, wherein the nonionic water-soluble group comprises a polyalkylene glycol. Clause 16. The probe according to Clause 15, wherein the polyalkylene glycol comprises polyethylene glycol. Clause 17. The probe according to any of the preceding clauses, wherein one or more acceptor fluorophores have an excitation maximum in the range of 450 to 750 nm. Clause 18. The probe according to Clause 17, wherein one or more acceptor fluorophores have excitation maxima in the range of 440-485 nm, 485-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-740 nm. Clause 19. The probe according to any one of Clauses 1 to 18, wherein one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm. Clause 20. The probe according to Clause 19, wherein one or more acceptor fluorophores have emission maxima in the range of 485-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 21. The probe according to any of the preceding clauses, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethine, dipyrometheneborone difluoride, naphthalimide, thiadin dyes, and acridine dyes. Clause 22. A probe as described in any of the preceding clauses, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores. Clause 23. The probe described in Clause 22, wherein the number of donor fluorophores is in the range of 2 to 20. Clause 24. The probe according to any of the preceding clauses, wherein the dye structure comprises a single acceptor fluorophore. Clause 25. The probe according to any of the preceding clauses, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, hydrocarbons, polyethylene glycol (PEG) polymers, lipids, peptoids, and polynucleotides. Clause 26. The probe according to Clause 25, wherein the non-conjugated polymer backbone comprises a peptide. Clause 27. The probe according to Clause 26, wherein the peptide comprises 5 to 100 amino acid residues. Clause 28. The probe according to any of the preceding clauses, wherein the quencher absorbs the light of the emission maximum of one or more acceptor fluorophores. Clause 29. The probe described in any of the preceding clauses, wherein the quencher absorbs light in the range of 450 to 875 nm. Clause 30. The probe according to Clause 29, wherein the quencher absorbs light in the range of 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 31. The probe described in Clause 29 or 30, wherein the quencher is selected from the Black Hole Quencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher. Clause 32. The probe according to Clause 31, wherein the quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), or tetramethylrhodamine (TAMRA). Clause 33. The probe according to any of the preceding clauses, wherein the dye structure is ligated to the 5' end of the nucleic acid and the quencher is ligated to the 3' end of the nucleic acid. Clause 34. The probe according to any one of Clauses 1 to 32, wherein the dye structure is ligated to the 3' end of the nucleic acid and the quencher is ligated to the 5' end of the nucleic acid. Clause 35. The probe according to Clause 33 or 34, further comprising a second quencher ligated to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid. Clause 36. The probe according to any one of Clauses 33 to 35, wherein the second quencher is ligated to a nucleotide located 9 or 10 nucleotides away from the 5' end of the nucleic acid. Clause 37. The probe according to Clause 35 or 36, wherein the second quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphoramido quencher, azoquencher, or anthraquinone quencher. Clause 38. A panel of probes for nucleic acid detection, A first nucleic acid complementary to the first target sequence, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A first dye structure linked to the first end of the first nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. A first pigment structure including; and A quencher ligated to the second end of the first nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; A first nucleic acid probe containing, A second nucleic acid complementary to the second target sequence, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A second dye structure linked to the first end of the second nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and A second dye structure comprising one or more acceptor fluorophores linked to the non-conjugated polymer backbone, which are in an energy transfer relationship with the donor fluorophore; and A quencher ligated to the second end of the second nucleic acid, which is in an energy transfer relationship with the acceptor fluorophore; A second nucleic acid probe containing, Includes, The first target sequence is different from the second target sequence, A panel in which the first dye structure has a different emission maximum than the second dye structure. Clause 39. The panel described in Clause 38, wherein the first and second nucleic acids consist of one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA). Clause 40. The panel according to Clause 38 or 39, wherein the nucleoside associated with each nucleotide in the plurality of nucleotides in each of the first and second nucleic acids is selected from canonical nucleosides, modified nucleosides, and nucleoside analogs. Clause 41. The panel according to Clause 40, wherein at least one nucleoside in the plurality of nucleotides in each of the first and second nucleic acids is a nucleoside analog. Clause 42. A panel according to any one of Clauses 38 to 41, further comprising one or more of the first nucleic acid probe and the second nucleic acid probe, with a secondary groove binder (MGB) linked thereto. Clause 43. The panel according to Clause 42, wherein the MGB is ligated to the 3' end of one or more of the first nucleic acid probe and the second nucleic acid probe. Clause 44. A panel according to any one of Clauses 38 to 43, wherein each of the first and second nucleic acids comprises 10 to 50 nucleotides. Clause 45. The panel according to Clause 44, wherein each of the first and second nucleic acids comprises 18 to 30 nucleotides. Clause 46. The panel according to any one of Clauses 38 to 45, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum in the range of 300 to 700 nm. Clause 47. A panel according to any one of Clauses 38 to 46, wherein one or more excitable donor fluorophores associated with the first nucleic acid probe and one or more excitable donor fluorophores associated with the second nucleic acid probe have different excitation maxima. Clause 48. The panel according to Clause 46 or 47, wherein one or more excitable donor fluorophores have excitation maxima in the range of 300-380 nm, 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-700 nm. Clause 49. The panel according to any one of Clauses 46 to 48, wherein one or more excitable donor fluorophores have emission maxima in the range of 380–440 nm, 440–485 nm, 485 nm–510 nm, 510–565 nm, 565–590 nm, 590–625 nm, or 625–725 nm. Clause 50. The panel according to any one of Clauses 38 to 49, wherein the one or more excitable donor fluorophores are selected from the group consisting of bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores. Clause 51. The panel according to any one of Clauses 38 to 50, wherein one or more excitable donor fluorophores are bonded to a water-soluble group. Clause 52. The panel according to Clause 51, wherein the water-soluble group comprises a nonionic water-soluble group. Clause 53. The panel according to Clause 52, wherein the nonionic water-soluble group comprises a polyalkylene glycol. Clause 54. The panel according to Clause 53, wherein the polyalkylene glycol comprises polyethylene glycol. Clause 55. The panel according to any one of Clauses 38 to 54, wherein one or more acceptor fluorophores have an excitation maximum in the range of 450 to 750 nm. Clause 56. A panel according to any one of Clauses 38 to 55, wherein one or more acceptor fluorophores associated with the first nucleic acid probe and one or more acceptor fluorophores associated with the second nucleic acid probe have different emission maxima. Clause 57. The panel according to Clause 56, wherein one or more acceptor fluorophores have excitation maxima in the range of 440–485 nm, 485–510 nm, 510–565 nm, 565–590 nm, 590–625 nm, or 625–740 nm. Clause 58. The panel according to any one of Clauses 38 to 57, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethine, dipyrometheneborone difluoride, naphthalimide, thiadin dyes, and acridine dyes. Clause 59. A panel according to any of Clauses 38 to 58, wherein the number of donor fluorophores in each of the first and second nucleic acid probes exceeds the number of acceptor fluorophores. Clause 60. The panel described in Clause 59, wherein the number of donor fluorophores is in the range of 2 to 20. Clause 61. The panel according to Clause 60, wherein the first pigment structure contains a different amount of donor fluorophore than the second pigment structure. Clause 62. A panel according to any one of Clauses 38 to 61, wherein the first and second dye structures each comprise a single acceptor fluorophore. Clause 63. A panel according to any one of Clauses 38 to 61, wherein the first dye structure comprises a different amount of acceptor fluorophores than the second dye structure. Clause 64. The panel according to any one of Clauses 38 to 63, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptoids, and polynucleotides. Clause 65. The panel according to Clause 64, wherein each of the non-conjugated polymer backbones comprises a peptide. Clause 66. The panel described in Clause 65, wherein each peptide contains 5 to 100 amino acid residues. Clause 67. The panel according to any one of Clauses 38 to 66, wherein the quencher absorbs the light of the emission maximum of one or more acceptor fluorophores. Clause 68. A panel according to any one of Clauses 38 to 67, wherein the quencher absorbs light in the range of 450 to 875 nm. Clause 69. The panel according to Clause 68, wherein the quencher absorbs light in the range of 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 70. The panel as described in any of Clauses 38-69, wherein the quencher is selected from Black Hole Quencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher. Clause 71. A panel according to any one of Clauses 38 to 70, wherein the quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), or tetramethylrhodamine (TAMRA). Clause 72. A panel according to any one of Clauses 38 to 71, wherein the first and second dye structures are ligated to the 5' ends of the first and second nucleic acids, and the quencher is ligated to the 3' ends of the first and second nucleic acids. Clause 73. A panel according to any one of Clauses 38 to 72, wherein the first and second dye structures are ligated to the 3' ends of the first and second nucleic acids, and the quencher is ligated to the 5' ends of the first and second nucleic acids. Clause 74. The panel according to Clause 72 or 73, further comprising a second quencher in which one or both of the first and second nucleic acid probes are ligated to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid. Clause 75. The panel according to any one of Clauses 72 to 74, wherein the second quencher is ligated to a nucleotide located 9 or 10 nucleotides away from the 5' end of the nucleic acid. Clause 76. The panel according to Clause 74 or 75, wherein the second quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphoramidite quencher, azoquencher, or anthraquinone quencher. Article 77. A kit for nucleic acid detection, A first nucleic acid complementary to the first target sequence, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A first dye structure linked to the first end of the first nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. A first pigment structure including; and A quencher ligated to the second end of the first nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; A kit for nucleic acid detection, including a first nucleic acid probe. Clause 78. A second nucleic acid complementary to the second target sequence, containing multiple nucleotides forming a chain from the 5' end to the 3' end; A second dye structure linked to the first end of the second nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. A second pigment structure including; and A quencher ligated to the second end of the second nucleic acid, which is in an energy transfer relationship with the acceptor fluorophore; Further comprising a second nucleic acid probe containing, The first target sequence is different from the second target sequence, The first dye structure has a different emission maximum than the second dye structure. The kit described in Article 77. Clause 79. The kit according to Clause 77 or 78, wherein the first and second nucleic acids consist of one or more deoxyribonucleic acid (DNA) and locked nucleic acid (LNA). Clause 80. The kit according to any one of Clauses 77-79, wherein the base associated with each nucleotide in the plurality of nucleotides in each of the first and second nucleic acids is selected from canonical bases, modified bases, and base analogs. Clause 81. The kit according to Clause 80, wherein at least one nucleotide among the plurality of nucleotides in each of the first and second nucleic acids is a nucleotide analog. Clause 82. A kit according to any one of Clauses 77 to 81, wherein at least one nucleic acid probe further comprises a minor groove binder (MGB). Clause 83. The kit according to Clause 82, wherein the MGB is ligated to the 3' end of the nucleic acid probe. Clause 84. A kit according to any one of Clauses 77 to 83, wherein each of the first and second nucleic acids comprises 10 to 50 nucleotides. Clause 85. The kit according to Clause 84, wherein each of the first and second nucleic acids comprises 18 to 30 nucleotides. Clause 86. The kit according to any one of Clauses 77 to 85, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum in the range of 300 to 700 nm. Clause 87. The kit according to any one of Clauses 77 to 86, wherein one or more excitable donor fluorophores associated with the first nucleic acid probe and one or more excitable donor fluorophores associated with the second nucleic acid probe have different excitation maxima. Clause 88. The kit according to Clause 86 or 87, wherein one or more excitable donor fluorophores have emission maxima in the range of 300-380 nm, 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-700 nm. Clause 89. The kit according to Clause 88, wherein one or more excitable donor fluorophores have emission maxima in the range of 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-725 nm. Clause 90. The kit according to any one of Clauses 77 to 89, wherein the one or more excitable donor fluorophores are selected from the group consisting of bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores. Clause 91. The kit according to any one of Clauses 77 to 90, wherein one or more excitable donor fluorophores are bonded to a water-soluble group. Clause 92. The kit according to Clause 91, wherein the water-soluble group comprises a nonionic water-soluble group. Clause 93. The kit according to Clause 92, wherein the nonionic water-soluble group comprises a polyalkylene glycol. Clause 94. The kit according to Clause 93, wherein the polyalkylene glycol comprises polyethylene glycol. Clause 95. The kit according to any one of Clauses 77 to 94, wherein one or more acceptor fluorophores have an excitation maximum in the range of 450 to 750 nm. Clause 96. A kit according to any one of Clauses 77 to 95, wherein one or more acceptor fluorophores associated with the first nucleic acid probe and one or more acceptor fluorophores associated with the second nucleic acid probe have different emission maxima. Clause 97. The kit according to Clause 95 or 96, wherein one or more acceptor fluorophores have excitation maxima in the range of 440–485 nm, 485–510 nm, 510–565 nm, 565–590 nm, 590–625 nm, or 625–740 nm. Clause 98. The kit according to any one of Clauses 77 to 97, wherein one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm. Clause 99. The kit according to Clause 98, wherein one or more acceptor fluorophores have emission maxima in the range of 485–510 nm, 510–565 nm, 565–590 nm, 590–625 nm, 625–740 nm, or 740–875 nm. Clause 100. The kit according to any one of Clauses 77 to 99, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethine, dipyrometheneborone difluoride, naphthalimide, thiadin dyes, and acridine dyes. Clause 101. The kit according to any of Clauses 77 to 100, wherein the number of donor fluorophores in each of the first and second nucleic acid probes exceeds the number of acceptor fluorophores. Clause 102. The kit described in Clause 101, wherein the number of donor fluorophores is in the range of 2 to 20. Clause 103. The kit according to any one of Clauses 77 to 102, wherein the first and second dye structures each comprise a single acceptor fluorophore. Clause 104. The kit according to any one of Clauses 77 to 103, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptoids, and polynucleotides. Clause 105. The kit according to Clause 104, wherein each of the non-conjugated polymer skeletons comprises a peptide. Clause 106. The kit according to Clause 105, wherein the peptide comprises 5 to 100 amino acid residues. Clause 107. The kit according to any one of Clauses 77 to 106, wherein the quencher absorbs the light of the emission maximum of one or more acceptor fluorophores. Clause 108. The kit according to any of Clauses 77 to 107, wherein the quencher absorbs light in the range of 450 to 875 nm. Clause 109. The kit according to Clause 108, wherein the quencher absorbs light in the range of 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 110. A kit as described in any of Clauses 77-109, wherein the quencher is selected from Black Hole Quencher (BHQ), QSY Quencher, Iowa Black Quencher, or QXL Quencher. Clause 111. The kit according to Clauses 77-110, wherein the quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), or tetramethylrhodamine (TAMRA). Clause 112. The kit according to any one of Clauses 77 to 111, wherein the first and second dye structures are ligated to the 5' ends of the first and second nucleic acids, and the quencher is ligated to the 3' ends of the first and second nucleic acids. Clause 113. The kit according to any one of Clauses 77 to 111, wherein the first and second dye structures are ligated to the 3' ends of the first and second nucleic acids, and the quencher is ligated to the 5' ends of the first and second nucleic acids. Clause 114. The kit according to Clause 112 or 113, further comprising a second quencher in which one or both of the first and second nucleic acid probes are ligated to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid. Clause 115. The kit according to any one of Clauses 112-114, wherein the second quencher is ligated to a nucleotide located 9 or 10 nucleotides away from the 5' end of the nucleic acid. Clause 116. The kit according to Clause 114 or 115, wherein the second quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphoramido quencher, azoquencher, or anthraquinone quencher. Clause 117. A kit according to any of Clauses 77 to 116, further comprising one or more of the following: buffer, water, deoxynucleotide triphosphate, nucleotide triphosphate, polymerase, 5'-3' nuclease, and polymerase having 5'-3' nuclease activity. Clause 118. A method for assaying a sample for the presence of a target nucleic acid, (a) preparing a reaction mixture, The reacting mixture is The aforementioned sample and, A nucleic acid complementary to the target nucleic acid, comprising multiple nucleotides that form a chain from the 5' end to the 3' end; A dye structure linked to the first end of the nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. Pigment structures including; and A quencher ligated to the second end of the nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; Nucleic acid probes and Includes, The nucleic acid probe hybridizes with any copy of the target nucleic acid in the sample, and (b) In order to assay the sample for the presence of the target nucleic acid, monitor the reaction mixture for the signal released from the dye structure, A method for assaying a sample for the presence of a target nucleic acid, including the target nucleic acid. Clause 119. The method according to Clause 118, further comprising irradiating the reaction mixture with the excitation wavelength of one or more donor fluorophores. Clause 120. The method according to any one of Clauses 118 to 119, wherein the reaction mixture further comprises a polymerase having 5'-3' nuclease activity, and monitoring the sample further comprises performing a polymerase chain reaction (PCR) on the reaction mixture. Article 121. The PCR is, A pair of amplification primers and deoxynucleotide triphosphates are provided to the reaction mixture. The first amplification primer contains a sequence complementary to a region of one strand of the target nucleic acid and can prime the synthesis of the complementary strand. The second primer contains a sequence complementary to the region of the second strand of the target nucleic acid and can prime the synthesis of the complementary strand. A polymerase exhibiting 5'-3' nuclease activity amplifies the target nucleic acid between the amplification primers. The nucleic acid probe is complementary to the region of the target nucleic acid located between the amplification primer pair, and The reaction temperature of the sample is cycled to a first temperature suitable for denaturing the target nucleic acid, then to a second temperature suitable for enabling hybridization of the amplification primer pair and the nucleic acid probe to the target nucleic acid, and then to a third temperature suitable for the polymerase to amplify the target nucleic acid. The method described in Clause 120, including the method described in Clause 120. Clause 122. The method according to Clause 121, further comprising monitoring the sample and repeating the cycle of the reaction temperature at least four times. Clause 123. The method according to any one of Clauses 118 to 119, further comprising the reaction mixture further comprising a polymerase having 5'-3' nuclease activity, monitoring the sample, and carrying out an isothermal amplification reaction. Clause 124. The method according to Clause 123, wherein the isothermal amplification reaction utilizes one or more of the following: nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand substitution amplification (SDA), recombinase polymerase amplification (RPA), and rolling circle amplification (RCA). Clause 125. The reaction mixture further comprises a second nucleic acid probe, The second nucleic acid probe described above, A second nucleic acid complementary to the second target nucleic acid, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A second dye structure linked to the first end of the second nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. A second pigment structure including; and A quencher ligated to the second end of the nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; Includes, The aforementioned sample contains or is suspected to contain a second target nucleic acid. The aforementioned target nucleic acid differs from the second target nucleic acid, The aforementioned dye structure has a different emission maximum than the second dye structure. The method described in any of clauses 118 to 124. Clause 126. The method according to any one of Clauses 118 to 125, wherein the first and second nucleic acids consist of one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA). Clause 127. The method according to any one of Clauses 118 to 126, wherein the base associated with each nucleotide in the plurality of nucleotides in each of the first and second nucleic acids is selected from canonical bases, modified bases, and base analogs. Clause 128. The method according to Clause 127, wherein at least one nucleotide among the plurality of nucleotides in each of the first and second nucleic acids is a nucleotide analog. Clause 129. The method according to any one of Clauses 118 to 128, wherein at least one nucleic acid probe further comprises a minor groove binder (MGB). Clause 130. The method of Clause 129, wherein the MGB is linked to the 3' end of the nucleic acid probe. Clause 131. The method of any one of Clauses 118 to 130, wherein the plurality of nucleotides in each of the first and second nucleic acids comprises 10 to 50 nucleotides. Clause 132. The method of Clause 131, wherein the plurality of nucleotides in each of the first and second nucleic acids comprises 18 to 30 nucleotides. Clause 133. The method of any one of Clauses 118 to 132, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum in the range of 300 to 700 nm. Clause 134. The method of any one of Clauses 118 to 133, wherein the one or more excitable donor fluorophores associated with the first nucleic acid probe and the one or more excitable donor fluorophores associated with the second nucleic acid probe have different excitation maxima. Clause 135. The method of Clause 133 or 134, wherein the one or more excitable donor fluorophores have an excitation maximum in the range of 300 to 380 nm, 380 to 440 nm, 440 to 485 nm, 485 nm to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, or 625 to 700 nm. Clause 136. The method of Clause 135, wherein the one or more excitable donor fluorophores have an emission maximum in the range of 380 to 440 nm, 440 to 485 nm, 485 nm to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, or 625 to 725 nm. Clause 137. The method of any one of Clauses 118 to 136, wherein the one or more excitable donor fluorophores are selected from the group consisting of bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores. Clause 138. The method of any one of Clauses 118 to 137, wherein the one or more excitable donor fluorophores are bound to a water-solubilizing group. Clause 139. The method according to Clause 138, wherein the water-solubilizing group comprises a nonionic water-solubilizing group. Clause 140. The method according to Clause 139, wherein the nonionic water-solubilizing group comprises a polyalkylene glycol. Clause 141. The method according to Clause 140, wherein the polyalkylene glycol comprises polyethylene glycol. Clause 142. The method according to any one of Clauses 118 to 141, wherein the one or more acceptor fluorophores have an excitation maximum in the range of 450 to 750 nm. Clause 143. The method according to any one of Clauses 118 to 142, wherein the one or more acceptor fluorophores associated with the first nucleic acid probe and the one or more acceptor fluorophores associated with the second nucleic acid probe have different emission maxima. Clause 144. The method according to Clause 142 or 143, wherein the one or more acceptor fluorophores have an excitation maximum in the range of 440 to 485 nm, 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, or 625 to 740 nm. Clause 145. The method according to any one of Clauses 118 to 144, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethines, dipyrromethene boron difluoride, naphthalimide, thiazine dyes, and acridine dyes. Clause 146. The method according to any one of Clauses 118 to 145, wherein the number of donor fluorophores in each of the first and second nucleic acid probes exceeds the number of acceptor fluorophores. Clause 147. The method according to Clause 146, wherein the number of donor fluorophores is in the range of 2 to 20. Clause 148. The method according to any one of Clauses 118 to 147, wherein the first and second dye structures each comprise a single acceptor fluorophore. Clause 149. The method according to any one of Clauses 118 to 148, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptoids, and polynucleotides. Clause 150. The method according to Clause 149, wherein each of the non-conjugated polymer skeletons comprises a peptide. Clause 151. The method according to Clause 150, wherein the peptide comprises 5 to 100 amino acid residues. Clause 152. The method according to any one of Clauses 118 to 151, wherein the quencher absorbs the light of the emission maximum of one or more acceptor fluorophores. Clause 153. The method according to any one of Clauses 118 to 152, wherein the quencher absorbs light in the range of 450 to 875 nm. Clause 154. The method according to Clause 153, wherein the quencher absorbs light in the range of 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 155. The method according to any one of Clauses 118 to 154, wherein the quencher is selected from Black Hole Quencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher. Clause 156. The method according to any one of Clauses 118 to 155, wherein the quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), or tetramethylrhodamine (TAMRA). Clause 157. The method according to any one of Clauses 118 to 156, wherein the first and second dye structures are ligated to the 5' ends of the first and second nucleic acids, and the quencher is ligated to the 3' ends of the first and second nucleic acids. Clause 158. The method according to any one of Clauses 118 to 156, wherein the first and second dye structures are ligated to the 3' ends of the first and second nucleic acids, and the quencher is ligated to the 5' ends of the first and second nucleic acids. Clause 159. The method according to Clause 157 or 158, further comprising a second quencher in which one or both of the first and second nucleic acid probes are ligated to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid. Clause 160. The method according to any one of Clauses 157 to 159, wherein the second quencher is ligated to a nucleotide located 9 or 10 nucleotides away from the 5' end of the nucleic acid. Clause 161. The method according to Clause 159 or 160, wherein the second quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphoramidite quencher, azoquencher, or anthraquinone quencher. Clause 162. The method according to any one of Clauses 118 to 161, wherein the reaction mixture further comprises one or more of the following: buffer, water, deoxynucleotide triphosphate, nucleotide triphosphate, polymerase, 5'-3' nuclease, and polymerase having 5'-3' nuclease activity. Article 163. Nucleic acids, Each comprises a first nucleic acid and a second nucleic acid, each containing multiple nucleotides that form a chain from the 5' end to the 3' end. The first nucleic acid is complementary to at least a portion of the second nucleic acid, The first nucleic acid described above is A dye structure linked to the first end of the first nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. Pigment structures including; and A quencher ligated to the second end of the first nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; Nucleic acids containing these substances. Clause 164. The nucleic acid described in Clause 163, wherein the first nucleic acid comprises one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA). Clause 165. The nucleic acid according to Clause 163 or 164, wherein the base associated with each nucleotide in the plurality of nucleotides of the first nucleic acid is selected from canonical bases, modified bases and base analogs. Clause 166. The nucleic acid according to Clause 165, wherein at least one nucleotide among the plurality of nucleotides is a nucleotide analog. Clause 167. The nucleic acid according to any one of Clauses 163 to 166, wherein the first nucleic acid further comprises a secondary groove binder (MGB) linked to the first nucleic acid. Clause 168. The nucleic acid according to Clause 167, wherein the MGB is ligated to the 3' end of the first nucleic acid. Clause 169. The nucleic acid according to any one of Clauses 163 to 168, wherein the plurality of nucleotides comprises 10 to 50 nucleotides. Clause 170. The nucleic acid according to Clause 169, wherein the plurality of nucleotides comprises 18 to 30 nucleotides. Clause 171. The nucleic acid according to any one of Clauses 163 to 170, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum in the range of 300 to 700 nm. Clause 172. The nucleic acid according to Clause 171, wherein one or more excitable donor fluorophores have excitation maxima in the range of 300-380 nm, 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-700 nm. Clause 173. The nucleic acid according to Clause 171 or 172, wherein one or more excitable donor fluorophores have emission maxima in the range of 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-725 nm. Clause 174. The nucleic acid according to any one of Clauses 163 to 173, wherein the one or more excitable donor fluorophores are selected from the group consisting of bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores. Clause 175. The nucleic acid according to any one of Clauses 163 to 174, wherein one or more excitable donor fluorophores are bonded to a water-soluble group. Clause 176. The nucleic acid according to Clause 175, wherein the water-soluble group comprises a nonionic water-soluble group. Clause 177. The nucleic acid according to Clause 176, wherein the nonionic water-soluble group comprises a polyalkylene glycol. Clause 178. The nucleic acid according to Clause 177, wherein the polyalkylene glycol comprises polyethylene glycol. Clause 179. The nucleic acid according to any one of Clauses 163 to 178, wherein one or more acceptor fluorophores have an excitation maximum in the range of 450 to 750 nm. Clause 180. The nucleic acid according to Clause 179, wherein one or more acceptor fluorophores have excitation maxima in the range of 440-485 nm, 485-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-740 nm. Clause 181. The nucleic acid according to any one of Clauses 163 to 180, wherein one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm. Clause 182. The nucleic acid according to Clause 181, wherein one or more acceptor fluorophores have emission maxima in the range of 485-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 183. The nucleic acid according to any one of Clauses 163 to 182, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethine, dipyrometheneborone difluoride, naphthalimide, thiadin dyes, and acridine dyes. Clause 184. A nucleic acid as described in any of Clauses 163 to 183, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores. Clause 185. The nucleic acid described in Clause 184, wherein the number of donor fluorophores is in the range of 2 to 20. Clause 186. The nucleic acid according to any one of Clauses 163 to 185, wherein the dye structure comprises a single acceptor fluorophore. Clause 187. The nucleic acid according to any one of Clauses 163 to 186, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptoids, and polynucleotides. Clause 188. The nucleic acid according to Clause 187, wherein the non-conjugated polymer backbone comprises a peptide. Clause 189. The nucleic acid according to Clause 188, wherein the peptide comprises 5 to 100 amino acid residues. Clause 190. The nucleic acid according to any one of Clauses 163 to 189, wherein the quencher absorbs the light of the emission maximum of one or more acceptor fluorophores. Clause 191. The nucleic acid according to any of Clauses 163 to 190, wherein the quencher absorbs light in the range of 450 to 875 nm. Clause 192. The nucleic acid according to Clause 191, wherein the quencher absorbs light in the range of 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 193. The nucleic acid described in any of Clauses 191 to 192, wherein the quencher is selected from Black Hole Quencher (BHQ), QSY quencher, Iowa Black Quencher, or QXL quencher. Clause 194. The nucleic acid according to Clause 193, wherein the quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), or tetramethylrhodamine (TAMRA). Clause 195. The nucleic acid according to any one of clauses 163 to 194, wherein the dye structure is linked to the 5' end of the first nucleic acid, and the quencher is linked to the 3' end of the first nucleic acid. Clause 196. The nucleic acid according to any one of clauses 163 to 194, wherein the dye structure is linked to the 3' end of the first nucleic acid, and the quencher is linked to the 5' end of the first nucleic acid. Clause 197. The nucleic acid according to clause 195 or 196, further comprising a second quencher linked to a nucleotide located 7 to 15 nucleotides away from the 5' end of the first nucleic acid. Clause 198. The nucleic acid according to any one of clauses 195 to 197, wherein the second quencher is linked to a nucleotide located 9 or 10 nucleotides away from the 5' end of the first nucleic acid. Clause 199. The nucleic acid according to clause 197 or 198, wherein the second quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphoramidite quencher, azo quencher, or anthraquinone quencher. Clause 200. A system for detecting a nucleic acid, comprising: a chamber for holding a sample; an illumination source directed to impinge on the sample; and a detector directed such that luminescence from the sample impinges on the detector; , wherein the sample comprises a nucleic acid probe, the nucleic acid probe comprises: a nucleic acid complementary to a target nucleic acid, comprising a plurality of nucleotides forming a strand from the 5' end to the 3' end; a dye structure linked to the first end of the nucleic acid, a non-conjugated polymer backbone, one or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. Pigment structures including; and A quencher ligated to the second end of the nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; A system for detecting nucleic acids, which contain or are suspected to contain the target nucleic acid, in the aforementioned sample. Clause 201. The system according to Clause 200, wherein the chamber includes a heating element for changing the temperature of the sample. Clause 202. The system according to Clause 201, wherein the heating element includes a water bath or a heating block. Clause 203. The system according to Clause 200 or 201, wherein the heating element utilizes a thermoelectric effect to change the temperature of the sample. Clause 204. The system described in any of Clauses 200-203, further including a user interface that enables the selection of temperature, illumination wavelength, detection wavelength, and temperature cycle program. Clause 205. The system according to any one of Clauses 200 to 204, wherein the nucleic acid comprises one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA). Clause 206. The system according to any one of Clauses 200 to 205, wherein the base associated with each nucleotide in the plurality of nucleotides is selected from canonical bases, modified bases, and base analogs. Clause 207. The system according to Clause 206, wherein at least one nucleotide among the plurality of nucleotides is a nucleotide analog. Clause 208. The system according to any one of Clauses 200 to 207, wherein the nucleic acid probe further comprises a sub-groove binder (MGB) linked to the nucleic acid probe. Clause 209. The system according to Clause 208, wherein the MGB is ligated to the 3' end of the nucleic acid probe. Clause 210. The system according to any one of Clauses 200 to 209, wherein the plurality of nucleotides comprises 10 to 50 nucleotides. Clause 211. The system according to Clause 210, wherein the plurality of nucleotides comprises 18 to 30 nucleotides. Clause 212. The system according to any one of Clauses 200 to 211, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum in the range of 300 to 700 nm. Clause 213. The system according to Clause 212, wherein one or more excitable donor fluorophores have excitation maxima in the range of 300-380 nm, 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-700 nm. Clause 214. The system according to Clause 212 or 213, wherein one or more excitable donor fluorophores have emission maxima in the range of 380-440 nm, 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, or 625-725 nm. Clause 215. The system according to any one of Clauses 200 to 214, wherein the one or more excitable donor fluorophores are selected from the group consisting of bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores. Clause 216. The system according to any one of Clauses 200 to 215, wherein one or more excitable donor fluorophores are bonded to a water-soluble group. Clause 217. The system according to Clause 216, wherein the water-soluble group comprises a nonionic water-soluble group. Clause 218. The system according to Clause 217, wherein the nonionic water-soluble group comprises a polyalkylene glycol. Clause 219. The system according to Clause 218, wherein the polyalkylene glycol comprises polyethylene glycol. Clause 220. The system according to any one of Clauses 200 to 219, wherein one or more acceptor fluorophores have an excitation maximum in the range of 450 to 750 nm. Clause 221. The system according to Clause 220, wherein one or more acceptor fluorophores have excitation maxima in the range of 440–485 nm, 485–510 nm, 510–565 nm, 565–590 nm, 590–625 nm, or 625–740 nm. Clause 222. The system according to any one of Clauses 200 to 221, wherein one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm. Clause 223. The system according to Clause 222, wherein one or more acceptor fluorophores have emission maxima in the range of 485-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 224. The system according to any one of Clauses 200 to 223, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethine, dipyrometheneborone difluoride, naphthalimide, thiadin dyes, and acridine dyes. Clause 225. A system as described in any of Clauses 200-224, in which the number of donor fluorophores exceeds the number of acceptor fluorophores. Clause 226. The system described in Clause 225, wherein the number of donor fluorophores is in the range of 2 to 20. Clause 227. The system according to any one of Clauses 200 to 226, wherein the dye structure comprises a single acceptor fluorophore. Clause 228. The system according to any one of Clauses 200 to 227, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptoids, and polynucleotides. Clause 229. The system according to Clause 228, wherein the non-conjugated polymer backbone comprises a peptide. Clause 230. The system according to Clause 229, wherein the peptide comprises 5 to 100 amino acid residues. Clause 231. The system according to any one of Clauses 200 to 230, wherein the quencher absorbs the light of the emission maximum of one or more acceptor fluorophores. Clause 232. The system according to any one of Clauses 200 to 231, wherein the quencher absorbs light in the range of 450 to 875 nm. Clause 233. The system according to Clause 232, wherein the quencher absorbs light in the range of 440-485 nm, 485 nm-510 nm, 510-565 nm, 565-590 nm, 590-625 nm, 625-740 nm, or 740-875 nm. Clause 234. The system described in Clause 232 or 233, wherein the quencher is selected from Black Hole Quencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher. Clause 235. The system according to Clause 234, wherein the quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), or tetramethylrhodamine (TAMRA). Clause 236. The system according to any one of Clauses 200 to 235, wherein the dye structure is ligated to the 5' end of the nucleic acid and the quencher is ligated to the 3' end of the nucleic acid. Clause 237. The system according to any one of Clauses 200 to 235, wherein the dye structure is ligated to the 3' end of the nucleic acid and the quencher is ligated to the 5' end of the nucleic acid. Clause 238. The system according to Clause 236 or 237, further comprising a second quencher ligated to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid. Clause 239. The system according to any one of Clauses 236 to 238, wherein the second quencher is ligated to a nucleotide located 9 or 10 nucleotides away from the 5' end of the nucleic acid. Clause 240. The system according to Clause 238 or 239, wherein the second quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, Iowa Black FQ, Iowa Black RQ, QXL490, QXL570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphoramido quencher, azoquencher, or anthraquinone quencher.

[0154] In at least some of the embodiments described above, one or more elements used in the embodiments may be interchangeably used in other embodiments, provided that such substitution is not technically impossible. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0155] In general, the terms used herein, and especially in the attached claims (e.g., the body of the attached claims), will be understood by those skilled in the art to be generally intended as “open” terms (for example, “including” should be interpreted as “including but not limited to,” “having” as “having at least,” and “includes” as “includes but not limited to,” and so on). Where a particular number is intended with respect to the description of an introduced claim, such intent will be explicitly stated in the claim, and where there is no such statement, such intent will be understood by those skilled in the art to be absent. For example, to aid understanding, the attached claims below may include the use of the introductory phrases “at least one” and “one or more” to introduce the description of a claim. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim description by the indefinite article "a" or "an" limits any particular claim containing such introduced claim description to only one embodiment containing such description, even if the same claim contains the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of the definite article used to introduce a claim description.Furthermore, even if a specific number is explicitly stated in relation to the description of an introduced claim, a person skilled in the art will recognize that such a statement should be interpreted as meaning at least the number stated (for example, the bare statement “two statements” without other modifiers means at least two statements, or two or more statements). Furthermore, when idiomatic expressions similar to “at least one of A, B, and C, etc.” are used, the construction is generally intended to be such that a person skilled in the art will understand the idiomatic expression (for example, “a system having at least one of A, B, and C” includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system having both A and B, a system having both A and C, a system having both B and C, and / or a system having both A, B, and C). When idiomatic expressions similar to “at least one of A, B, or C” are used, it is generally intended that they be constructed in a sense that a person skilled in the art would understand (for example, “a system having at least one of A, B, or C” includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system having both A and B, a system having both A and C, a system having both B and C, and / or a system having both A, B, and C). A person skilled in the art will further understand that substantially any disjunctive word and / or phrase presenting two or more alternative terms should be understood as construing the possibility of including one of the terms, either of the terms, or both of the terms, whether in the specification, claims, or drawings. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B”.

[0156] Furthermore, if any feature or aspect of this disclosure is described in relation to the Markush group, a person skilled in the art will likely recognize that this disclosure also describes any individual member or subgroup of a member of the Markush group.

[0157] As will be understood by those skilled in the art, for all purposes, for the benefit of providing, for example, written explanations, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. Any scope enumerated can be readily recognized as sufficiently explainable and enabling the division of the same scope into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can readily be divided into a lower third, a middle third, an upper third, etc. Also, as will be understood by those skilled in the art, all phrases such as “up to,” “at least,” “greater than,” and “less than” include the number stated and refer to a scope that can be subsequently divided into subscopes as discussed above. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group containing 1 to 5 items refers to a group containing 1, 2, 3, 4, or 5 items, and so on.

[0158] While the aforementioned inventions are described in some detail as examples and illustrations for clarity, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of the present invention without departing from the spirit or scope of the appended claims.

[0159] Therefore, the foregoing merely illustrates the principles of the present invention. Those skilled in the art will understand that various configurations embodying the principles of the present invention and falling within its spirit and scope can be devised, although not expressly described or shown herein. Furthermore, all examples and conditional statements described herein are intended primarily to help the reader understand the principles of the present invention and the concepts to which the inventors have contributed to advancing the art, and should be construed as not being limited to such specifically described examples and conditions. Moreover, all descriptions herein describing the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. In addition, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any developed elements that perform the same function regardless of their structure. Furthermore, nothing disclosed herein is intended to be made available to the public, whether such disclosure is expressly described in the claims or not.

[0160] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims. In the claims, § 112(f) or § 112(6) of the U.S. Patent Act is expressly defined as being invoked for the limitation in a claim only when the exact phrase “means for” or the exact phrase “step for” is found at the beginning of such limitation in the claim, and if such exact phrase is not used in the limitation in the claim, § 112(f) or § 112(6) of the U.S. Patent Act is not invoked.

Claims

1. A probe for nucleic acid detection, A nucleic acid complementary to the target sequence, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A dye structure linked to the first end of the nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. Pigment structures including; and A quencher ligated to the second end of the nucleic acid, which is in an energy transfer relationship with one or more acceptor fluorophores; A probe for nucleic acid detection, including [specific component].

2. The probe according to claim 1, wherein the nucleic acid is composed of one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA).

3. The probe according to claim 1 or 2, wherein the base associated with each nucleotide in the plurality of nucleotides is selected from canonical bases, modified bases, and base analogs.

4. The probe according to claim 3, wherein at least one nucleotide among the plurality of nucleotides is a nucleotide analog.

5. The probe according to any of the preceding claims, wherein the plurality of nucleotides comprises 10 to 50 nucleotides.

6. The probe according to claim 5, wherein the plurality of nucleotides comprises 18 to 30 nucleotides.

7. A probe according to any of the preceding claims, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

8. The probe according to any of the preceding claims, wherein the dye structure comprises a single acceptor fluorophore.

9. The probe according to any of the preceding claims, wherein the non-conjugated polymer backbone comprises at least one of a peptide, a carbohydrate, a hydrocarbon, a polyethylene glycol (PEG) polymer, a lipid, a peptoid, and a polynucleotide.

10. The probe according to claim 9, wherein the non-conjugated polymer skeleton comprises a peptide.

11. A panel of probes for nucleic acid detection, A first nucleic acid complementary to the first target sequence, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A first dye structure linked to the first end of the first nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. A first pigment structure including; and A quencher ligated to the second end of the first nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; A first nucleic acid probe containing, A second nucleic acid complementary to the second target sequence, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A second dye structure linked to the first end of the second nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. A second pigment structure including; and A quencher ligated to the second end of the second nucleic acid, which is in an energy transfer relationship with the acceptor fluorophore; A second nucleic acid probe containing, Includes, The first target sequence is different from the second target sequence, A panel in which the first dye structure has a different emission maximum than the second dye structure.

12. A kit for nucleic acid detection, A first nucleic acid complementary to the first target sequence, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A first dye structure linked to the first end of the first nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. A first pigment structure including; and A quencher ligated to the second end of the first nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; A kit for nucleic acid detection, including a first nucleic acid probe.

13. A method for assaying a sample for the presence of a target nucleic acid, (a) preparing a reaction mixture, The reacting mixture is The aforementioned sample and, A nucleic acid complementary to the target nucleic acid, comprising multiple nucleotides that form a chain from the 5' end to the 3' end; A dye structure linked to the first end of the nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. Pigment structures including; and A quencher ligated to the second end of the nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; Nucleic acid probes and Includes, The nucleic acid probe hybridizes with any copy of the target nucleic acid in the sample, and (b) In order to assay the sample for the presence of the target nucleic acid, monitor the reaction mixture for the signal released from the dye structure, A method for assaying a sample for the presence of a target nucleic acid, including the target nucleic acid.

14. nucleic acids, It comprises a first nucleic acid and a second nucleic acid, each containing multiple nucleotides that form a chain from the 5' end to the 3' end, The first nucleic acid is complementary to at least a portion of the second nucleic acid; The first nucleic acid described above is A dye structure linked to the first end of the first nucleic acid, Non-conjugated polymer skeleton, One or more donor fluorophores linked to the non-conjugated polymer backbone, and One or more acceptor fluorophores linked to the non-conjugated polymer backbone are in an energy transfer relationship with the donor fluorophore. Pigment structures including; and A quencher ligated to the second end of the first nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; Nucleic acids containing these substances.

15. A system for detecting nucleic acids, A chamber for holding the sample; A light source directed to collide with the aforementioned sample; and A detector directed so that the light emitted from the aforementioned sample collides with the detector; Includes, The aforementioned sample contains a nucleic acid probe, The nucleic acid probe, A nucleic acid complementary to the target nucleic acid, containing multiple nucleotides that form a chain from the 5' end to the 3' end; A dye structure linked to the first end of the nucleic acid, A non-conjugated polymer skeleton, one or more donor fluorophores linked to the non-conjugated polymer skeleton, and one or more acceptor fluorophores linked to the non-conjugated polymer skeleton that are in an energy transfer relationship with the donor fluorophores. Pigment structures including; and A quencher ligated to the second end of the nucleic acid, which is in an energy transfer relationship with an acceptor fluorophore; Includes, A system for detecting nucleic acids, which contain or are suspected to contain the target nucleic acid, in the aforementioned sample.

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