Scaffolded chromophores for nucleic acid detection and methods and uses thereof

Abstract

Provided herein are scaffolded chromophores and systems, methods, and uses thereof for nucleic acid detection. Certain embodiments relate to a nucleic acid probe comprising a nucleic acid complementary to a target sequence. The probe further comprises a dye structure attached to a first end of the nucleic acid and comprises a non-conjugated polymeric scaffold having one or more donor fluorophores and one or more acceptor fluorophores attached thereto, wherein the donor fluorophores and the acceptor fluorophores are in an energy transfer relationship. Such a probe can further comprise a quencher attached to a second end of the nucleic acid, wherein the quencher and the one or more acceptor fluorophores are in an energy transfer relationship. Other embodiments include a second nucleic acid probe comprising a second nucleic acid complementary to a different target sequence.

Description

Cross-references to related applications

[0001] This application claims priority to the filing date of U.S. Provisional Patent Application Serial No. 63 / 539,067, filed on September 18, 2023, the disclosure of which is incorporated herein by reference. Background Technology

[0002] Nucleic acid testing has wide applications in scientific and medical fields such as diagnostics and / or environmental monitoring. However, these tests rely on fluorescently labeled probes, which may have certain limitations, including low sensitivity, weak fluorescence intensity, or inability to achieve multiplexing. Given these limitations, there is a need for probes that can be tuned to enhance fluorescence emission and / or multiplex. Summary of the Invention

[0003] This document provides scaffold-type chromophores for nucleic acid detection, as well as systems, methods, and uses thereof. Some embodiments involve nucleic acid probes comprising a nucleic acid complementary to a target sequence. The probe also includes a dye structure attached to a first end of the nucleic acid and a non-conjugated polymer backbone having one or more donor fluorophores and one or more acceptor fluorophores attached thereto, wherein the donor and acceptor fluorophores are in an energy transfer relationship. Such probes may also include a quencher attached to a second end of the nucleic acid, wherein the quencher and one or more acceptor fluorophores are in an energy transfer relationship. Other embodiments include a second nucleic acid probe comprising a second nucleic acid complementary to a different target sequence.

[0004] The probes in some implementations may include DNA, RNA, and / or locked nucleic acids. Individual bases may include standard bases, modified bases, base analogs, minor groove binding agents, water-soluble groups, and / or additional quenchers. The non-conjugated polymer backbone may consist of one or more of peptides, carbohydrates, hydrocarbons, polyethylene glycol polymers, lipids, peptide-like molecules, and polynucleotides.

[0005] Other embodiments include methods, systems, kits, and hybridization products comprising nucleic acid probes as described above. Such methods can be used to detect target nucleic acids and include generating a reaction mixture comprising a sample and a nucleic acid probe, and monitoring signals emitted by dye structures in the reaction mixture to determine the presence of the target nucleic acid in the sample. Such methods may include performing nucleic acid amplification reactions, such as PCR and / or isothermal amplification. Kits may contain probes as described above and one or more reagents for performing reactions (such as nucleic acid amplification reactions). Other embodiments involve products comprising probes that hybridize with target nucleic acids as described above.

[0006] The system includes a sample chamber for holding the sample, an illumination source oriented to irradiate the sample, and a detector such that light emitted from the sample illuminates the detector. Other systems may include heating elements, such as heating blocks and / or water baths. Other systems may include a user interface to allow selection of temperature, illumination wavelength, detection wavelength, and temperature cycling programs. Attached Figure Description

[0007] A better understanding of the invention can be achieved by reading the following detailed description in conjunction with the accompanying drawings. The drawings include the following figures: Figure 1 Illustrative nucleic acid probes according to various embodiments of the present invention are provided, the probes having a scaffold-like chromophore dye structure and a quencher.

[0008] Figure 2 Non-limiting examples of nucleic acid probes according to various embodiments of the present invention are provided, the probes having a scaffold-like chromophore dye structure and a quencher.

[0009] definition As used herein, the terms “chemoselective functional group” and “chemoselective tag” are used interchangeably to refer to a functional group that, under certain circumstances, can selectively react with another compatible functional group to form a covalent bond after optionally activating one of its functional groups. Chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their active esters, and groups that can react with each other via click chemistry, such as azides and alkynyl groups (e.g., cyclooctyrynyl), tetrazides, trans-cyclooctene, dienes and diene affinities, as well as azides, sulfonyl(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, and hydroxyl, hydrazide, hydrazine, aldehyde, ketone, azide, alkyne, phosphine, epoxide, etc.

[0010] As used herein, the term "sample" refers to material or mixture of materials containing one or more analytes of interest (e.g., nucleic acids), in some cases in liquid form. In some embodiments, when used in its broadest sense, the term refers to any plant, animal, or bacterial material containing cells or producing cellular metabolites, such as tissues or fluids isolated from an individual (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or tissues or fluids isolated from components of in vitro cell cultures, as well as samples from the environment. The term "sample" can also refer to a "biological sample." As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells, or components (e.g., bodily fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to a homogenate, lysate, or extract prepared from a whole organism or a subset or fraction thereof of its tissues, cells, or components, including but not limited to plasma, serum, cerebrospinal fluid, lymph, external skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. In some embodiments, the sample has been taken from an animal or plant. Biological samples may include cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of a living organism (both eukaryotes and prokaryotes) having at least a nucleus and a cell membrane. In some embodiments, cells include prokaryotic cells, such as prokaryotic cells from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants, or fungi. The term "sample" can also refer to "environmental sample." As used herein, the term "environmental sample" refers to a swab collected from a non-biological location (e.g., a high-frequency contact location, including but not limited to door handles, toilet seats, faucets, water dispensers, etc.). "Environmental samples" can also refer to liquids (e.g., water, beverages, fuels, petroleum residues), gases (e.g., air, medical oxygen, industrial oxygen, industrial nitrogen), soil, filter membranes, 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 any polymer of nucleotides of any length, including nucleic acids of 2-100 nucleotides and those longer than 50 nucleotides. The term "nucleotide" refers to a sugar, a base, and a phosphate group. In this document, "nucleobase" and "base" are used interchangeably. The term "nucleic acid" includes polymers of standard bases (adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)) and non-standard bases, chemically or biochemically modified or derivatized nucleotides, and nucleotides with modified sugar-phosphate backbones in which the conventional backbone has been replaced by a non-natural or synthetic backbone. Conventional backbones are generally considered to be ribose-phosphate backbones (as used in ribonucleic acid (RNA)) and deoxyribose-phosphate backbones (as used in deoxyribonucleic acid (DNA)). Non-natural, synthetic, or other unconventional backbones include those in which ribose or deoxyribose is replaced by another sugar (e.g., threonine), peptide, or other components. Examples of non-naturally occurring, synthetic, or other unconventional nucleic acid backbones include xenobiotic nucleic acids (XNA), peptide nucleic acids (PNA), morpholinonucleotides, locked nucleic acids (LNA), glycol nucleic acids (GNA), 1,5-anhydrohexyl alcohol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), fluoroarabinonucleotides (FANA), and threononucleotides (TNA). Some nucleic acids may contain one or more nucleotides with unconventional backbones in addition to the conventional backbone; for example, one or more nucleotides may be LNA nucleotides, while the remaining nucleotides are DNA nucleotides. Nucleic acids can have any convenient length, such as 2 or more nucleotides, 4 or more nucleotides, 10 or more nucleotides, 20 or more nucleotides, 50 or more nucleotides, 100 or more nucleotides, 300 or more nucleotides, or up to 500 or 1000 or more nucleotides.

[0012] As used herein, the term "separated" refers to a fraction of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, or even at least 99% free of other components associated with the fraction prior to purification.

[0013] "Multiple" contains at least two members. In some cases, multiple can have five or more members, such as 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, ten thousand or more, one hundred thousand or more, one hundred thousand or more, one hundred thousand or more.

[0014] The number range includes the numbers that define the range.

[0015] The term "specific binding" refers to the direct association between two molecules, such as through covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions like salt bridges and water bridges. Specific binding members describe the members of a pair of molecules that have a specific binding affinity to each other. Members of specific binding pairs can be naturally derived or wholly or partially synthesized.

[0016] Specific binding members can be nucleic acids. In nucleic acids, a specific nucleotide can pair with one or more other nucleotides, such as classical pairing (adenine-thymine, cytosine-guanine) or ambiguous pairing (e.g., inosine-cytosine, inosine-adenine, etc.). Nucleic acids typically pair with their anticomplementary sequences, but some specific binding pairs allow for ambiguous pairings or mismatches. Therefore, the members of this pair have a specific property of binding to each other. Such affinity or binding force can often be expressed by its melting temperature (T). m The term "affinity" is used to measure the strength of binding, with higher melting temperatures indicating stronger binding or higher affinity between nucleic acid pairs. "Affinity" refers to the strength of the binding, and increased binding affinity is associated with higher melting temperatures.

[0017] As used herein, the term "hybridization conditions" refers to the conditions under which a primer or other polynucleotide specifically hybridizes with a region of the target nucleic acid that is complementary to it. Whether a primer specifically hybridizes with the target nucleic acid depends on several factors, such as the degree of complementarity between the polymer and the target nucleic acid, and the temperature at which hybridization occurs, which can be determined by the primer's melting temperature (T0). M The melting temperature refers to the temperature at which one half of the primer-target nucleic acid duplex remains hybridized while the other half dissociates into single strands. (The duplex...) Tm The value can be determined experimentally or predicted using the following formula: Tm = 81.5 + 16.6(log10[Na+]) + 0.41 (G+C ratio) – (60 / N), where N is the chain length and [Na+] is less than 1 M. 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 based on various parameters can also be used to predict primer / target duplexes depending on different hybridization conditions. TmMethods for achieving specific nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Chapter 2, Part 1, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier (1993).

[0018] As used herein, the terms “complementarity” and “complementarity” refer to nucleotide sequences that form base pairs with all or part of a target nucleic acid (e.g., a region of the product nucleic acid) through non-covalent bonds. In classic Watson-Crick base pairing, adenine (A) pairs with thymine (T), just as guanine (G) pairs with cytosine (C) in DNA. In RNA, thymine (T) is replaced by uracil (U). Therefore, A is complementary to T, and G is complementary to C. In RNA, A is complementary to U, and vice versa. Generally, “complementarity” refers to nucleotide sequences that are at least partially complementary. The term “complementarity” can also include completely complementary duplexes, such that every nucleotide in one strand is complementary to every nucleotide at a corresponding position in the other strand. In some cases, a nucleotide sequence may be partially complementary to the target, and not all nucleotides may be complementary to every nucleotide at every corresponding position in the target nucleic acid. For example, primers can be completely complementary to the target nucleic acid (i.e., 100%), or primers and target nucleic acids can have a certain degree of complementarity, but not complete complementarity (e.g., 70%, 75%, 85%, 90%, 95%, 99%).

[0019] The percentage of identity between two nucleotide sequences can be determined by sequence alignment to achieve optimal alignment (e.g., by introducing gaps in the first sequence to achieve optimal alignment). The nucleotides at corresponding positions are then compared, and the percentage of identity between the two sequences is a function of the number of identical positions shared by these sequences (i.e., percentage of identity = number of identical positions / total number of positions × 100). A molecule is identical at that position when a position in one sequence is occupied by the same nucleotide as the corresponding position in another sequence. A non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Integration of such algorithms into the NBLAST and XBLAST programs (version 2.0) is described in Altschul et al., NucleicAcids Res. 25:389-3402 (1997). When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. In one respect, the parameters for sequence comparison can be set to fraction=100 and word length=12, or the parameters can be changed (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), then “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), at which the intensity of emitted radiation (e.g., light) is highest.

[0021] The method described herein may include multiple steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as needed. Therefore, the time between each step may be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer, and may include 5 hours or longer. In some embodiments, each subsequent step is performed immediately after the completion of the preceding step. In other embodiments, the steps may be performed after an incubation or waiting period following the completion of the preceding step, which may be, for example, a waiting period ranging from several minutes to overnight.

[0022] As used herein, the terms “assessment,” “determination,” “measurement,” “evaluation,” and “determination” are used interchangeably and include both quantitative and qualitative determinations.

[0023] The term "joint" or "linker" refers to a connecting portion that links two groups and has a main chain of 100 atoms or less in length. A joint or linker can be a covalent bond connecting the two groups or a chain of 1 to 100 atoms in length, such as a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more carbon atoms, wherein the joint can be straight, branched, cyclic, or monoatomic. In some cases, the joint is a branched joint, which refers to a connecting portion that links three or more groups. In some cases, one, two, three, four, or five or more carbon atoms in the joint main chain may optionally be substituted with sulfur, nitrogen, or oxygen heteroatoms. In some cases, the joint main chain includes connecting functional groups such as ethers, thioethers, amino groups, amides, sulfonamides, carbamates, thiocarbamates, ureas, thioureas, esters, thioesters, or imines. The bonds between the main chain atoms can be saturated or unsaturated, and in some cases, the linker main chain contains no more than one, two, or three unsaturated bonds. The linker can contain one or more substituents, such as alkyl, aryl, or alkenyl groups. Linkers can include, but are not limited to, polyethylene glycol; ethers, thioethers, tertiary amines, and alkyl groups, which can be straight-chain or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker main chain can include cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, wherein two or more atoms of the cyclic group, for example, two, three, or four atoms, are contained in the main chain. The linker can be cleavable or incleavable.

[0024] As used herein, the terms “water-soluble group,” “water-soluble group,” and “WSG” are used interchangeably and refer to a group or substituent that is readily solubilized in an aqueous environment, such as under physiological conditions, and imparts improved water solubility to the attached molecule. WSG can increase the solubility of probe, dye structures, or other components thereof (e.g., donor or acceptor fluorophores) in predominantly aqueous solutions compared to a control dye structure or its components lacking WSG. The water-soluble group can be any convenient hydrophilic group that is readily solubilized in an aqueous environment.

[0025] Various water-soluble polymer groups are suitable for use in the WSG of the subject dyes. Any convenient water-soluble group (WSG) can be included in the dyes described herein to provide greater water solubility. While the increase in solubility may vary, in some cases the increase (compared to compounds without WSG) is 2 times or more, such as 5 times, 10 times, 25 times, 50 times, 100 times or more. In some cases, the hydrophilic water-soluble group is charged, for example, positively or negatively charged. In some cases, the hydrophilic water-soluble group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., as described herein). In some cases, the WSG is linear. In some embodiments, the WSG is a hydrophilic polymer, such as polyethylene glycol, modified PEG, peptide sequences, peptide-like substances, carbohydrates, oxazoline, polyols, dendritic polymers, dendritic polyglycerol, cellulose, chitosan, or derivatives thereof. Water-soluble groups of interest include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonates, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, hydroxyl groups, amines, amino acids, ammonium, guanidine salts, pyridinium, polyamines and sulfonium, polyols, linear or cyclic sugars, primary amines, secondary amines, tertiary or quaternary amines and polyamines, phosphonate groups, phosphonite groups, ascorbate groups, glycols including polyethers, -COOM′, -SO3M′, -PO3M′, -NR3 + Y′, (CH2CH2O) p R and mixtures thereof, wherein Y′ can be any halogen, sulfate, sulfonate or oxyanion, p can be 1 to 500, each R can be independently H or alkyl (e.g. methyl) and M′ can be a cationic counterion or hydrogen, --(CH2CH2O). yy CH2CH2XR yy --(CH2CH2O) yy CH2CH2X--、--X(CH2CH2O) yy CH2CH2--, ethylene glycol and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S and NR. ZZ And R ZZ and R YY Independently selected from H and C 1-3 Alkyl group. In some cases, WSG is (CH2). x (OCH2CH2) y OCH3, where each x is an integer independently from 0 to 20, and each y is an integer independently from 0 to 50. In some cases, the water-soluble groups include nonionic polymers (e.g., PEG polymers) whose ends are replaced with ionic groups (e.g., sulfonate groups).

[0026] In some embodiments of the formula, the pendant group of interest includes those selected from (CH2).x (OCH2CH2) y The substituents of OCH3, wherein each x is an integer from 0 to 20, and each y is an integer from 0 to 50; and the benzyl group is optionally replaced by one or more halogens, hydroxyl groups, C1-C... 12 Alkyl group or (OCH2CH2) z OCH3 substitution, where each z is an independent integer from 0 to 50. In some cases, the substituent is (CH2)3(OCH2CH2). 11 OCH3. In some embodiments, one or more substituents are benzyl groups substituted with at least one WSG group (e.g., one or two WSG groups) selected from (CH2). x (OCH2CH2) y OCH3, where each x is an integer from 0 to 20 independently, and each y is an integer from 0 to 50 independently.

[0027] Multiple WSGs can be included at a single position in the subject dye via a branching linker. In some embodiments, the branching linker is an aralkyl substituent further disubstituted with a water-soluble group. Thus, in some cases, the branching linker group is a substituent of the dye that links the dye to two or more water-soluble groups. In some embodiments, the branching linker is an amino acid, such as a lysine amino acid linked to three groups via an amino and a carboxylic acid group. In some cases, incorporating multiple WSGs via a branching linker imparts desired solubility to the dye. In some cases, the WSG is a nonionic side chain group capable of imparting water solubility exceeding 50 mg / mL. In some cases, the WSG is a nonionic side chain group capable of imparting water solubility exceeding 100 mg / mL. In some embodiments, the dye includes substituents selected from alkyl, aralkyl, and heterocyclic groups, each group further substituted with a hydrophilic polymer group including a water-soluble group, such as polyethylene glycol (PEG) (e.g., a PEG group with 6-24 units).

[0028] Water-soluble polymers of interest suitable for use with WSG include polyethylene glycol (PEG) groups or modified PEG groups. These water-soluble polymers include, but are not limited to, polyepoxide-based polymers, such as polyethylene glycol "PEG" (see, for example, "Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", JM Harris, Ed., Plenum Press, New York, NY (1992); and "Poly(ethyleneglycol) Chemistry and Biological Applications", JM Harris and S. Zalipsky, Eds.).ACS (1997); and International Patent Applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964Wamikokuryugo4,179,337;5,075,046;5,089,261;5,100,992;5,134,192;5,166,309;5,1 71,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,64 3,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,44 2; 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;6,214,966). .

[0029] Examples of water-soluble polymers of interest include, but are not limited to, those containing polyepoxides, polyamide epoxides, or derivatives thereof, including polyepoxides and polyamide epoxides comprising an ethylene oxide repeating unit of the formula -(CH2-CH2-O)-. Other examples of polymers of interest include those having the formula -[C(O)-XC(O)-NH-Y-NH]n- or -[NH-Y-NH-C(O)-XC(O)]. n - A polyamide with a molecular weight greater than 1,000 Daltons, wherein X and Y are divalent groups that may be the same or different and may be branched or linear, and n is a discrete integer from 2 to 100, such as 2 to 50, and wherein one or both of X and Y contain a biocompatible, substantially non-antigenic, water-soluble repeating unit, which may be linear or branched. Other examples of water-soluble repeating units include ethylene oxides of the formula -(CH2-CH2-O)- or -(O-CH2-CH2)-. The number of such water-soluble repeating units can vary significantly, ranging from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6 to 100, for example 2 to 50 or 6 to 50. An example of an embodiment is one in which one or both of X and Y are selected from: -((CH2) n1 -(CH2-CH2-O) n2 -(CH2)- or -((CH2) n1 -(O-CH2-CH2) n2 -(CH2) n-1 -), where n1 is 1 to 6, 1 to 5, 1 to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, or 2 to 5. A further example of an implementation is an example where X is -(CH2-CH2)-, and where 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 either end or both ends to include, for example, terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy, or substituted alkoxy, etc.) and / or terminal linking functional groups (e.g., amino or carboxylic acid groups suitable for linking via amide bonds) to a molecule of interest (e.g., via branching groups to a photocollecting chromophore). The subject water-soluble polymer can be adapted to include any convenient linking groups. It will be understood that in some cases, the water-soluble polymer may include some degree of dispersibility with respect to polymer length, depending on the methods used to prepare and / or purify the polymer starting materials. In some cases, the water-soluble polymer is monodisperse.

[0031] Water-soluble polymers may include one or more spacer groups or connectors. Examples of spacer groups or connectors include linear or branched moieties that contain one or more repeating units used in the water-soluble polymer, diamino and / or diacid units, natural or non-natural amino acids or derivatives thereof, and aliphatic moieties including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., which may contain, for example, up to 18 carbon atoms or even additional polymer chains.

[0032] The water-soluble polymer moiety or one or more spacer groups or linkers (if present) of the polymer moiety may comprise a biostable or biodegradable polymer chain or unit. For example, polymers with repeating linkers exhibit varying degrees of stability under physiological conditions, depending specifically on bond instability. Polymers with such bonds can be classified according to their relative hydrolysis rates under physiological conditions, based on known hydrolysis rates, for example, from less unstable to more stable (e.g., polyurethane (-NH-C(O)-O-) > polyorthoester (-OC((OR)(R'))-O-) > polyamide (-C(O)-NH)-). Similarly, linker systems that connect water-soluble polymers to target molecules can be biostable or biodegradable, for example, from less unstable to more stable: carbonate (-OC(O)-O-) > ester (-C(O)-O-) > urethane (-NH-C(O)-O-) > orthoester (-OC((OR)(R'))-O-) > amide (-C(O)-NH-). Generally, given the instability of sulfate groups, the use of sulfated polysaccharides may be avoided. Furthermore, the use of polycarbonates and polyesters may be less desirable. These bonds are provided as examples only and are not intended to limit the types of bonds that can be used in the polymer chains or linker systems of the water-soluble polymers available in the WSGs disclosed herein.

[0033] Water-soluble groups (WSGs) can endow subject probe or dye structures with water solubility exceeding 10 mg / mL, such as exceeding 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. In some cases, branched nonionic water-soluble groups (WSGs) can endow subject probe or dye structures with solubility of 20 mg / mL or greater in water (e.g., aqueous buffers), such as 30 mg / mL or greater, 40 mg / mL or greater, 50 mg / mL or greater, 60 mg / mL or greater, 70 mg / mL or greater, 80 mg / mL or greater, 90 mg / mL or greater, 100 mg / mL or greater, or even more. It is understood that dipyrrole methylene-based water-soluble dyes form discrete, water-solubilized nanoparticles in aqueous systems under certain conditions. In some cases, water-solubilized nanoparticles can be resistant to aggregation and can be used for a variety of bioassays.

[0034] The terms "polyethylene oxide," "PEO," "polyethylene glycol," and "PEG" are used interchangeably and refer to substances containing the formula --(CH2----O--). n - The polymeric group of the described chain or a derivative thereof. In some embodiments, "n" is 5000 or less, such as 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, such as 3 to 15, or 10 to 15. It should be understood that the PEG polymeric group can have any convenient length and can include a variety of terminal groups and / or additional substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide terminal and / or substituents. PEG groups applicable to subject polychromatophores include those described by S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165 and by Zhu et al. in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112 (8), pp 4687–4735.

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

[0036] The term "substituted alkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms in the alkyl chain have optionally been replaced by heteroatoms (such as -O-, -N-, -S-), -S(O). n - (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) substituted and having 1 to 5 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclicoxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclicoxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NR a R b , where R ’ and R ” They may be the same or different, and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0037] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. For example, alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, etc. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and ynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and ynyl are as defined herein.

[0038] The term “substituted alkoxy” refers to a substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- group, wherein the substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0039] "Alkenyl" refers to a monovalent, branched or linear, cyclic or acyclic hydrocarbon group containing a carbon-carbon double bond. Exemplary alkenyl groups include vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenoenyl, and 24-carbonenyl. In some cases, alkenyl groups contain 1 to 24 carbon atoms, such as 1 to 18 carbon atoms or 1 to 12 carbon atoms. The term "lower alkenyl" refers to an alkyl group having 1 to 6 carbon atoms.

[0040] "Alynyl" or "alkynyl group" refers to a straight-chain or branched monovalent hydrocarbon group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1, preferably 1 to 2, triple bond unsaturation sites. Examples of such alkynyl groups include ethynyl (-C≡CH) and propynyl (-CH2C≡CH).

[0041] The term “substituted alkynyl” or “substituted alkyne” refers 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, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0042] "Heterocyclic group" refers to a single radical cyclic group that contains a heteroatom (e.g., O, S, N) as a ring atom and is not aromatic (i.e., distinguishing heterocyclic groups from heteroaryl groups). Exemplary heterocyclic groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocyano.

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

[0044] "Aryl" itself, or as part of another substituent, refers to a monovalent aromatic hydrocarbon group derived by removing a hydrogen atom from a single carbon atom in an aromatic ring system. Aryl groups of interest include, but are not limited to, groups derived from anthracene, acenaphthene, phenanthrene, anthracene, azulene, benzene, benzoxene, fluorene, phenanthrene, hexane, hexalene, asymmetric indarene, symmetric indarene, indene, indene, naphthalene, octaphenyl, octane, cyclooctatetraene, oleophane, pentadiene, pentaphenyl, cyclopentadiene, pentanene, perylene, phenanthracene, phenanthrene, septene, pyrene, pinanthracene, rubiginium, benzo[9,10]phenanthrene, trinaphthalene, etc. In some embodiments, the aryl group comprises 6 to 20 carbon atoms. In some embodiments, the aryl group comprises 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0045] "Substituted aryl" means, unless otherwise defined by the aryl substituent, an aryl group substituted with 1 to 5 or 1 to 3 substituents selected from acyloxy, hydroxyl, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, amide, alkylaryl, aryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, aminoacyloxy, oxyamide, 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] "Heteroaryl" itself, or as part of another substituent, refers to a monovalent heteroaryl derived by removing a hydrogen atom from a single atom in a heteroaryl ring system. Heteroaryl groups of interest include, but are not limited to, groups derived from acridine, arsenoindene, carbazole, β-carboline, chromene, chromene, zoline, furan, imidazole, indazole, indole, dihydroindole, indoleazine, isobenzofuran, isochromene, isoindole, isoindoleline, isoquinoline, isothiazole, isoxazole, naphthidine, oxadiazole, oxazole, phenidine, phenanthril, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrroleazine, quinazoline, quinoline, quinazine, quinoxaline, tetrazolium, thiadiazole, thiazolium, triazole, benzotriazole, thiophene, triazole, xanthan, benzodioxane, etc. In some embodiments, the heteroaryl group is derived from a 5-20 membered heteroaryl group. In some embodiments, the heteroaryl group is derived from a 5-10 membered heteroaryl group. In some embodiments, the heteroaryl group is derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0047] "Heterocyclic," "heterocyclic," "heterocyclic alkyl," and "heterocyclic group" refer to a saturated or unsaturated group having a monocyclic or multiple fused rings (including fused bridged rings and spirocyclic systems) and having 3 to 20 ring atoms (including 1 to 10 heteroatoms). These ring atoms are selected from nitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or more rings may be cycloalkyl, aryl, or heteroaryl, provided that the attachment point is through a non-aromatic ring. In some embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0048] Examples of heterocyclic and heteroaryl compounds include, but are not limited to, aza-butane, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indoleazine, isoindole, indole, dihydroindole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthroxaline, isothiazine, phenazine, isoxazine, phenoxazine, phenanthridine Thiazide, imidazoline, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazoline, thiophene, benzo[b]thiophene, morpholino, thiomorpholino (also known as thiozamorpholino), 1,1-dioxothiomorpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc.

[0049] "Substituted heteroaryl" means, unless otherwise defined, a heteroaryl group substituted with 1 to 5 or 1 to 3 substituents selected from acyloxy, hydroxyl, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, amide, alkylaryl, aryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, aminoacyloxy, oxyamide, 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 “alkylaryl” or “aryl” refer to the group -alkylene-aryl and substituted alkylene-aryl, wherein the alkylene, substituted alkylene and aryl are as defined herein.

[0051] "Alkylene" refers to a divalent aliphatic hydrocarbon group, preferably having 1 to 6, more preferably 1 to 3, carbon atoms, which is straight-chain or branched, and optionally composed of one or more carbon atoms selected from -O-, -NR-. 10 -、-NR 10 C(O)-、-C(O)NR 10 - and other group intervals. For example, the term includes methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), etc. "Substituted alkylene" refers to an alkylene in which one to three hydrogens are substituted with substituents as described for carbon in the definition of "substituted" below.

[0052] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by the same or different substituents. Substituents of interest include, but are not limited to, alkylene dioxy groups (such as methylene dioxy groups), -MR groups, etc. 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 Where M is a halogen; R 60 R 61 R 62 and R 63 Independently, it is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cyclohexaalkyl, substituted cyclohexaalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally, R 60 and R 61 Together with the nitrogen atoms they are bonded to, they form cyclohexaalkyl or substituted cyclohexaalkyl rings; and R 64 and R 65 Independently, it is hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cyclohexaalkyl, substituted cyclohexaalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R 64 and R 65 Together with the nitrogen atoms they are bonded to, they form cyclohexaalkyl or substituted cyclohexaalkyl rings. In some embodiments, the 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 In some implementations, substituents include -M and -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 - In some implementations, substituents include -M and -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 -, where R 60 R 61 and R 62 As defined above. For example, the substituted group may have a methylenedioxy substituent or one, two, or three substituents selected from halogen atoms, (1-4C) alkyl groups, and (1-4C) alkoxy groups. When the substituted group is an aryl or heteroaryl group, the substituent (e.g., as described herein) may be referred to as an "aryl substituent".

[0053] It should be understood that, of all the substituents defined above, polymers obtained by defining a substituent as having further substituents (e.g., a substituted aryl group having a substituted aryl group as a substituent, which itself is substituted by a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended to be included herein. In this case, the maximum number of such substitutions is three. For example, the successive substitutions of substituted aryl groups that are particularly considered herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

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

[0055] "Halogen" and "halogen" refer to chlorine, bromine, fluorine, and iodine groups.

[0056] "Carboxyl group", "carboxyl group" and "carboxylate" refer to the -CO2H group and its salt.

[0057] "Sulfonyl" refers to the group -SO2R, where R is an alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic group, or its substituted form. Exemplary sulfonyl groups include -SO2CH3 and -SO2(C6H5).

[0058] Unless otherwise stated, mentioning an atom means including all isotopes of that atom. For example, mentioning H means including... 1 H, 2 H (i.e., D) and 3 H (i.e. T), and mentioning C means including 12 All isotopes of C and carbon (e.g.) 13 C). Furthermore, any group described includes all stereoisomers of that group.

[0059] Unless otherwise stated, the nomenclature of substituents not explicitly defined herein is derived by naming the terminal portion of the functional group, followed by the adjacent functional group toward the junction. For example, the substituent “arylalkoxycarbonyl” refers to the group (aryl)-(alkyl)-OC(O)-.

[0060] For any group disclosed herein containing one or more substituents, it is understood that such group does not contain any sterically impractical and / or synthetically infeasible substitution or substitution patterns. Furthermore, the subject compounds include all stereochemical isomers resulting from the substitution of these compounds. Detailed Implementation

[0061] This document provides scaffold-type chromophores for nucleic acid detection, methods thereof, and uses. The scaffold-type chromophores of embodiments of the present invention comprise: a probe formed from a nucleic acid complementary to a target sequence; a dye structure attached to a first end of a nucleic acid comprising a non-conjugated polymer backbone; one or more excitable donor fluorophores attached to the non-conjugated polymer backbone; and one or more acceptor fluorophores attached to the non-conjugated polymer backbone, wherein the donor and acceptor fluorophores are in an energy transfer relationship; and a quencher attached to a second end of the nucleic acid, wherein the quencher and one or more acceptor fluorophores are in an energy transfer relationship. Methods for preparing and using the scaffold-type chromophores for nucleic acid detection are also provided, as well as kits comprising dyes and usable in method embodiments.

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

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Nevertheless, for clarity and ease of reference, certain terms are defined below. Furthermore, representative illustrative methods and materials are described herein, although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention.

[0064] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range (unless otherwise explicitly stated by the context, up to one-tenth of the lower limit unit) and any other stated value or intermediate value within the stated range are covered by this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered by this invention, but are subject to any specific exclusions within the stated range. When a stated range includes one or two limits, the range excluding one or both of the included limits is also included in this invention.

[0065] The numerical ranges given in this article are preceded by the term "approximately". The term "approximately" is used to provide literal support for the exact number that follows, as well as numbers that are close to or approximate to the number following the term. In determining whether a number is close to or approximates a specifically listed number, an unlisted number that is close to or approximates can be a number that provides a basic equivalent to the specifically listed number in the presented context.

[0066] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials relating to the publication reference. References to any publication refer to its disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to a prior invention prior to such publication. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0067] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a precondition for combining elements of the claim using exclusive terms such as “unique,” ​​“only,” or using a negative limiting term.

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

[0069] For the sake of grammatical fluency and functional interpretation, the apparatus and method have been or will be described. However, it should be clearly understood that, unless expressly stated in accordance with 35 USC §112, the claims should not be construed as necessarily being limited in any way by the interpretation of “apparatus” or “step”, but should be given the full meaning and scope of the definitions provided by the claims under the doctrine of equivalents, and where the claims are expressly stated in accordance with 35 USC §112, they should be given all statutory equivalents under 35 USC §112.

[0070] In further describing certain embodiments of the invention, a scaffold-based chromophore for nucleic acid detection is first reviewed in more detail, followed by a review of methods for using and preparing the dye, and a review of kits containing the scaffold-based chromophore for nucleic acid detection.

[0071] Scaffold-type chromophores for nucleic acid detection As described above, this disclosure provides scaffold-based chromophores for nucleic acid detection. As used herein, a "scaffold-based chromophore" refers to a chromophore, including a fluorophore, arranged in an energy transfer relationship (e.g., FRET). Scaffold-based chromophores include donor and acceptor chromophores arranged on a common structure (e.g., backbone). Detection can be qualitative (e.g., presence / absence), quantitative (e.g., amount of target), and / or semi-quantitative (e.g., relative amount of target). Scaffold-based chromophores can be used as probes in nucleic acid amplification reactions, including polymerase chain reaction (PCR) and its variants, 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 Figure 1 As shown, some embodiments involve a probe for identifying a target nucleic acid. To detect the target nucleic acid, some embodiments include a nucleic acid 102 complementary to the target sequence. In some embodiments, the nucleic acid is linked to a dye structure 104 and a quencher 106. Further details of each component will be discussed below.

[0073] Nucleic acid Nucleic acid 102 is composed of multiple nucleotides forming a chain from the 5' end to the 3' end. Nucleic acid 102 can have any length to hybridize with and / or be specific to a specific target sequence under specific conditions (such as the reaction conditions of an amplification reaction). The temperature at which hybridization with the target sequence is allowed can be determined by the melting temperature (or T) of the specific sequence. m To measure it. T m It can typically be measured and determined by various available methods. In some embodiments, nucleic acid 102 can have any convenient length, such as 2 or more nucleotides, like 4 or more nucleotides, 10 or more nucleotides, 20 or more nucleotides, 50 or more nucleotides, 100 or more nucleotides, 300 or more nucleotides, like up to 500 or 1000 or more nucleotides. Some embodiments include nucleic acids having 10-50 nucleotides. Some embodiments include nucleic acids having 18-30 nucleotides.

[0074] Nucleic acid 102 and / or individual nucleotides can be formed from polymers of standard bases (adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U)) and non-standard bases (also known as analogs), chemically or biochemically modified or derivatized nucleotides, and nucleotides with modified sugar-phosphate backbones (where the conventional backbone has been replaced by a non-natural or synthetic backbone).

[0075] Various nucleotide modifications and analogues are known in the art, including 1-methyl adenosine, 2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine, 2-methyl adenosine, 2-O-ribosylphosphoadenosine, N6-methyl-N6-threonylcarbamoyl adenosine, N6-acetyl adenosine, N6-glycylcarbamoyl adenosine, N6-isopentenyl adenosine, N6-methyl adenosine, N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, N6-(cis-hydroxyisopentenyl) adenosine, N6-hydroxyn-valinecarbamoyl adenosine, 1,2-O-dimethyl adenosine, N6,2-O-dimethyl adenosine, 2-O-methyl adenosine, N6,N6,O-2-trimethyl adenosine, 2-methylthio-N6-(cis-hydroxy) (Isopentenyl) adenosine, 2-methylthio-N6-methyl adenosine, 2-methylthio-N6-isopentenyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, 2-thiocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, lysine, 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-ribosylphosphoguanosine, 7-methylguanosine, modified hydroxyhuaitin (h ydroxywybutosine), 7-aminomethyl-7-denitroguanosine, 7-cyano-7-denitroguanosine, N2,N2-dimethylguanosine, 4-demethylglycine, epoxyguanosine, hydroxyguanosine, isoguanosine, 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, peroxyguanosine, galactosylguanosine, mannosylguanosine, guanosine, archipelone, guanosine, methylglycine, glycine, 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-taurylmethyluridine, 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, methyl uridine 5-oxyacetic acid, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurylmethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine, 2-O-methylpseudouridine, inosine, 1-methylinosine, 1,2-O-dimethylinosine, and 2-O-methylinosine. These can all be components of publicly available nucleic acids.

[0076] Conventional backbones are generally considered to be ribose-phosphate backbones (as used in ribonucleic acid (RNA)) and deoxyribose-phosphate backbones (as used in deoxyribonucleic acid (DNA)). Non-natural, synthetic, or other unconventional backbones include those where ribose or deoxyribose is replaced by another sugar (e.g., threon), peptide, or other moiety. Examples of non-natural, synthetic, or other unconventional nucleic acid backbones include xenobiotic nucleic acids (XNA), peptide nucleic acids (PNA), morpholinonucleotides, locked nucleic acids (LNA), glycol nucleic acids (GNA), 1,5-dehydrated hexadiol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), fluoroarabinonucleotides (FANA), and threononucleotides (TNA). Some nucleic acids may contain one or more nucleotides with unconventional backbones in addition to a conventional backbone; for example, one or more nucleotides may be LNA nucleotides, while the remaining nucleotides are DNA nucleotides.

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

[0078] polymer skeleton As described above, the dye structure of the embodiment includes a polymer backbone 108. In some cases, the polymer backbone 108 consists of non-conjugated repeating units having any convenient configuration (e.g., linear, branched, or dendritic polymer configuration). The polymer backbone 108 can be a linear polymer. The polymer backbone 108 can be branched. In some cases, the dye structure includes multiple dangling donor chromophores (e.g., donor fluorophore 110 and acceptor fluorophore 112), each dangling donor chromophore independently linked to a non-conjugated repeating unit of the polymer backbone. The configuration of the dangling groups can be achieved during or after the synthesis of the polymer backbone. The incorporation of dangling groups can be achieved in a sequence-specific manner, depending on the specific synthetic method used, by means of configuration, block configuration, or stepwise synthesis.

[0079] The term "unit" refers to a structural subunit of a polymer. The term "unit" is intended to include monomers, comonomers, coblocks, repeating units, etc. A "repeating unit" is a subunit of a polymer defined by the minimum number of unique structural features required for the unit to be considered a monomer, such that when the unit is repeated n times, the resulting structure describes the polymer or its blocks. In some cases, a polymer may include two or more distinct repeating units, for example, when the polymer is a multiblock polymer, a random arrangement of units, or a defined sequence, each block may define a different repeating unit. It should be understood that there are many ways to arrange repeating units or blocks, and in the formulas shown in the polymer backbone described herein, any convenient linear arrangement of various lengths can be included in the overall polymer structure. It should be understood that a polymer can also be represented by a formula expressing the mol% value of each unit in the polymer, and such a formula can represent many arrangements of repeating units, such as random or multiblock polymers or defined residue sequences. In some cases, the repeating unit of a polymer comprises a single monomer group. In some cases, the repeating unit of a polymer comprises two or more monomer groups, i.e., comonomer groups, such as two, three, four, or more comonomer groups. The term "comonomer" or "comonomer group" refers to a structural unit of a polymer, which may itself be part of a repeating unit 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 can fall in the range of 2 to 500,000, such as 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 such as 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 repeating units or segments in the skeleton may fall within the range of 2 to 1,000, such as 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 other cases, the specific number of repeating units or segments in the skeleton may fall within the range of 2 to 500, such as 2 to 400, 2 to 300, 2 to 200, or 2 to 100 units or segments. In still other cases, the specific number of repeating units or segments in the skeleton may fall within the range of 2 to 100 repeating units, such as 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 co-block configurations of the non-conjugated repeating units. Alternatively, the polymer backbone may include a specifically defined sequence of the non-conjugated repeating units, such as amino acid residues of a polypeptide sequence. These configurations may be characterized by polymer fragments of the repeating units (e.g., as described herein), which themselves may be repeated throughout the modular scaffold.

[0082] "Non-conjugated" means that at least a portion of the repeating unit includes a saturated main-chain group (e.g., a group having two or more consecutive monovalent bonds) that excludes π-conjugation or delocalized electronic structures extending along the polymer backbone from one repeating unit to the next. It should be understood that even if a repeating unit may not be conjugated with adjacent repeating units, such a repeating unit may also include one or more unsaturated groups individually comprising unsaturated bonds (e.g., alkenyl or alkynyl unsaturated bonds) and / or aryl or heteroaryl groups, which may be part of the backbone. In some cases, each repeating unit of the polymer backbone includes a side chain comprising a linked dangling group or a chemically selective tag for attachment to the dangling group.

[0083] In some embodiments of dye structure 104, polymer backbone 108 is a linear polymer. In some cases, the linear polymer is selected from peptides, peptide-like substances, hydrocarbon polymers, PEG polymers, carbohydrates, and polynucleotides. In some cases, the linear polymer is a peptide. In some cases, the linear polymer is a peptide-like substance. In some cases, the polymer is a hydrocarbon polymer. In some other cases, the polymer is a PEG polymer. Further details regarding polymer backbones that can be used in embodiments of the present invention can be found in PCT application serial numbers PCT / US2019 / 024662 (published as WO2019 / 191482) and PCT application serial number PCT / US2020 / 019510 (published as WO2020 / 222894); the disclosures of these applications are incorporated herein by reference.

[0084] In some cases, the dye structure comprises 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 comprises 2 or more amino acids, such as 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, up to a maximum of 100 amino acids. In some cases, the dye structure comprises 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 necessary, the polymer backbone present in the dye structure of the embodiments of the present invention may be replaced by one or more water-soluble groups (WSG), such as those defined above.

[0086] Donor fluorophore and acceptor fluorophore As described above, the dye structure of certain embodiments may include one or more donor fluorophores 110 and one or more acceptor fluorophores 112, all attached to the polymer backbone 108. In some embodiments, there is an energy transfer relationship between the donor fluorophores 110 and the acceptor fluorophores 112. An energy transfer relationship refers to the situation where the emission of the donor fluorophore 110 is absorbed by the acceptor fluorophore 112. In some cases, the emission maximum of the donor fluorophore 110 is located at the excitation (or absorption) maximum of the acceptor fluorophore 112. The number of donor fluorophores 110 may be 1 to 20, including 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 number of fluorophores between 1 and 20. In addition, the number of receptor fluorophores can be 1 to 20, such as 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or any integer number of fluorophores between 1 and 20.

[0087] By allowing various combinations or numbers of donor and acceptor fluorophores, certain implementations provide a tunable fluorescence mechanism for nucleic acid detection. This tunability can be achieved by varying the number of donor and / or acceptor fluorophores within the dye structure. In some cases, the light emission of the acceptor fluorophore can be proportional to the number of donor fluorophores, thus enhancing emission as the number of donor fluorophores increases. This process can be used to improve probe sensitivity, for example, allowing the detection of trace or extremely trace amounts of target sequences. For instance, to enhance acceptor fluorophore emission, the probe can contain more donor fluorophores, exceeding the number of acceptor fluorophores.

[0088] The combination of donor and acceptor fluorophores can be selected from fluorophores with known FRET relationships. In some cases, the excitation maxima of the donor fluorophore are approximately 300 nm to approximately 700 nm. These wavelengths include near-ultraviolet (near UV) to red light wavelengths. In some cases, the excitation maxima of the donor fluorophore can range from 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). Typically, the emission maximum is a wavelength greater than the excitation maximum, and depending on the excitation and Stokes shift of the specific fluorophore, the resulting emission maximum of the donor fluorophore according to certain embodiments may be 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. Exemplary chromophores include bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores. Such fluorophores are found in US patents 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, and 10,018. The disclosures are described in one or more of the following: U.S. Patent Publications 2023 / 0152327, 2022 / 0348770, 2021 / 0032474, and 2020 / 0225143; and U.S. Applications 18 / 201,940, 18 / 201,649, and 18 / 120,554; the disclosures of which are incorporated herein by reference in their entirety.

[0089] As described above, FRET utilizes acceptor fluorophores whose excitation distribution includes the emission maxima of selected donor fluorophores. In some embodiments, the emission maxima of the selected donor fluorophores are approximately equal to the excitation maxima of the acceptor fluorophores (e.g., ±25 nm). In some embodiments, the excitation maxima of one or more acceptor fluorophores range from 450 to 750 nm. Depending on the specific type of acceptor fluorophore, the desired emission maxima, and their Stokes shift, such maxima can include excitation maxima in the blue region (about 440 nm to about 485 nm), cyan region (about 485 nm to about 510 nm), green region (about 510 nm to about 565 nm), yellow region (about 565 nm to about 590 nm), orange region (about 590 nm to about 625 nm), or red region (about 625 nm to about 740 nm). Given an excitation wavelength and Stokes shift, the emission maximum of various acceptor fluorophores can be approximately 500 nm to 875 nm, typically falling within the cyan to near-infrared (near IR) light range, such as 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, xanthan, cyanine, polyacetylenes, pyrene, thiazide, acridine, dipyrrole methyl boron difluoride, naphthimide, phycobiliprotein, dinoflagellate chlorophyll, their conjugates, and combinations thereof. In some embodiments, the acceptor fluorophore (A) is a cyanine dye, xanthan dye, coumarin dye, thiazide dye, or acridine dye. In some cases, the receptor fluorophore (A) is selected from DY 431, DY 485XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831 and diethylaminocoumarin. Fluorescent dyes of interest include, but are not limited to, Texas Red, Cascade Blue, Cascade Yellow, coumarin, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (polydoxyphylla-chlorophyll-a protein), PerCP-Cy5.5, Lucifer Yellow, Marina Blue, Oregon Green 488, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, and BODIPY TR, its conjugates, and combinations thereof. Lanthanide chelates of interest include, but are not limited to, europium chelates, terbium chelates, and samarium chelates. In some embodiments, the polymer dye structure includes a polychromatic group linked to an acceptor fluorophore selected from Cy5, Cy5.5, Cy7, Alexa488, Alexa 647, and Alexa700.In some embodiments, the polymer dye includes a polychromatic group linked to an acceptor fluorophore selected from Dyomics dyes (e.g., DY 431, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY 831) and diethylaminocoumarin. In some cases, the receptor fluorophore (A) is selected from Texas Red, California Red, iFluor594, Cascade Blue, Cascade Yellow, coumarin, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight 350, DyLight 405, DyLight 488, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, phycoerythrin, PerCP (polydoxyphylla-chlorophyll-a protein), PerCP-Cy5.5, Lucifer Yellow, Marina Blue, Oregon Green 488, Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa... Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, their conjugates and combinations thereof.Various dyes are known in the art, and / or as described in U.S. Patent Nos. 11,668,716, 11,624,089, 11,407,773, 11,371,078, 11,214,688, 11,130,865, 11,119,107, 10,605,813, 10,228,375, 10,053,484, 9,863,947, 9,797,899, 9,719,998, 9,551,029, 9,493,656, 9,383,353, 9,228,225, 9,034,920, 8,753,814, 8,163,910, 8,0 The disclosures described in one or more of the following U.S. patent publications are incorporated herein by reference in their entirety: 30,096, 7,767,829, 7,704,284, 7,563,907, 7,402,671, 7,256,291, 7,038,063, 6,750,346, 6,743,568, 6,399,392, 6,225,050, 6,191,278, 6,048,982, 5,286,803, and 4,945,171; and U.S. Patent Publications 2020 / 0277670, 2011 / 010575, 2006 / 0199949, and 2003 / 0165942.

[0091] If necessary, the donor fluorophore and / or acceptor fluorophore present in the dye structure of the embodiments of the present invention may be replaced by one or more water-soluble groups (WSG), such as those defined above.

[0092] The dye structures that may 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 Application Publications Nos. 2022 / 0348770 and 2023 / 0152327; and pending U.S. Patent Application Serial Nos. 18 / 120,554, 18 / 127,568 and 18 / 201,940, the disclosures of which are incorporated herein by reference.

[0093] Quenching agent As described above, the nucleic acid probe may contain a quencher 106 that has an energy transfer relationship with one or more receptor fluorophores 112. The quencher 106 may be attached to one end of the nucleic acid 102 (e.g., the 5' end or the 3' end). In some embodiments, the quencher 106 is attached to the 3' end of the nucleic acid 102. Some embodiments include an internal quencher 106, wherein the quencher 106 is attached to a nucleotide not located at the end. The internal quencher may be located 7 to 15 nucleotides away from the dye structure (e.g., 7 to 15 nucleotides away from the 5' end of the nucleic acid). In some specific embodiments, the internal quencher is located 9 or 10 nucleotides away from the 5' end of the nucleic acid. The internal quencher may be used in conjunction with a terminal quencher (e.g., a quencher located at the 5' end or the 3' end), or may replace the terminal quencher.

[0094] The quencher can contain any convenient absorbing portion. The quencher can be a small molecule quencher. In some cases, the quencher is a fluorescent dye with an emission profile significantly longer than the emission maximum of the donor fluorophore (e.g., the aforementioned acceptor and / or donor fluorophores). In some embodiments, the quencher is a non-fluorescent portion. In some embodiments, the absorption and / or excitation profile of the quencher includes the emission maximum of the aforementioned acceptor fluorophore (e.g., about 500 nm to 875 nm). The absorption and / or excitation profile of the quencher may also include the emission maximum of the aforementioned donor fluorophore (e.g., about 450 nm to 725 nm). Therefore, the quencher can absorb light from about 450 nm to about 875 nm, including the ranges of 440 to 485 nm, 485 nm to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm, or 740 to 875 nm.

[0095] The quencher may be selected from Black Hole Quencher (BHQ), QSY quencher, Iowa Black, or QXL quencher. In some embodiments, the quencher is selected from QSY-7, QSY-9, QSY-21, QSY-35, BHQ-1, BHQ-2, BHQ-3, IowaBlack FQ, Iowa Black RQ, QXL 490, QXL 570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphorus amide quencher, azo quencher, anthraquinone quencher, conjugates thereof, and combinations thereof.

[0096] The specific quencher used can be selected based on the parameters of interest, including (but not limited to) the excitation maximum, the emission maximum, the Stokes shift, the extinction coefficient, and / or any other relevant factors.

[0097] Such quenchers are described in one or more of U.S. Patent Nos. 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.

[0098] Other aspects of the probe Some embodiments of the probe may include features that enhance binding or hybridization efficiency with the target sequence. Such modifications may include minor groove binding agents (MGBs). Some embodiments may include one or more, two or more, or any other number of MGBs on the probe. The MGB may be attached to the 5' or 3' end of the nucleic acid. For the purposes of this disclosure, a molecule is considered an MGB if it can bind to the minor groove of double-stranded DNA with a binding constant of 10⁻³ M⁻¹ or higher. Such binding can be detected by well-established spectrophotometric methods, such as ultraviolet spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, or by gel electrophoresis.

[0099] MGBs may contain certain naturally occurring compounds, such as fusiformin, terbinafine, lexitropsin, scintillan, chromomycin A3, oligomycin, amiodarone, siberiamycin, and many other related antibiotics and synthetic derivatives. Certain quaternary ammonium heterocyclic compounds, diarylamins such as pentamidine, stiptamidine, berenil, CC-1065, and related pyrroloindole and indole polypeptides, Hoechst 33258, 4′-6-diamidinyl-2-phenylindole (DAPI). Various oligopeptides composed of natural or synthetic amino acids are minor groove binding compounds, including 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylic acid ester (CDPI) and N-methylpyrrolo-4-carboxy-2-amide (MPC). Some embodiments include dimers, trimers, tetramers, and pentamers of CDPI. Other implementations include dimers, trimers, tetramers, and pentamers of MPC. Other examples of MGB can be found in U.S. Patent Nos. 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 into probe sets for multiplexed detection. In this case, the nucleic acid 102 of each probe is complementary to a different target sequence—for example, the first nucleic acid is complementary to the first target sequence, the second nucleic acid is complementary to the second target sequence, and the first target sequence is different from the second target sequence. The first and second target sequences can be located on the same molecule (e.g., a chromosome) or on different molecules. In some cases, the first and second target sequences are located within the same amplicon (e.g., a region amplified by a primer set). Furthermore, to distinguish probes in a group, each probe can use a different combination of donor and / or acceptor fluorophores. Some embodiments use the same donor fluorophore but use acceptor fluorophores with different emission wavelengths. This allows for simultaneous detection of probes using only one excitation source. Other simultaneous detection options may include donor fluorophores with similar excitation and emission distributions. Some embodiments have probes using donor and / or acceptor fluorophores with different excitation or emission distributions. In this case, excitation or detection can be performed asynchronously. Those skilled in the art will understand how to select donor and / or acceptor fluorophores and appropriate excitation and detection.

[0101] A probe set can contain as many probes as possible, which can be identified by simultaneous and / or asynchronous detection. For example, a limited number of fluorophores may have the same excitation maximum but different emission maximums, thus limiting the number of probes that can be detected simultaneously. Therefore, some sets may use multiple collections of such fluorophores so that a first excitation source can detect multiple probes, while a second excitation source can detect even more probes. Examples of such fluorophores can be found in U.S. Patent Applications Nos. 18 / 201,940 and 18 / 120,554, the disclosures of which are incorporated herein by reference in their entirety.

[0102] The number of different probes in a given group can vary. In some cases, the number is 2 or more, such as 5 or more, including 10 or more, such as 25 or more, 50 or more, 75 or more, etc. In other cases, the number can be 1000 or less, such as 500 or less, including 250 or less, such as 200 or less, 150 or less, including 100 or less. The number of probes in a group can 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. Probe groups can target specific gene sets, enabling the group to have diagnostic or prognostic capabilities for diseases such as cancer. Other groups may focus on the Recommended Uniform Screening Panel (RUSP). The RUSP is a list of conditions recommended by the U.S. Secretary of Health and Human Services as part of a universal newborn screening program. Some groups may target microbial traces in the sample, such as for diagnosing infectious pathogens and their variants (optional). For example, a group could be used to identify tuberculosis and certain antibiotic-resistant variants.

[0103] A set of probes can be added to a single reaction, such as a real-time PCR or quantitative PCR reaction. In some cases, the set is provided as a collection of individual probes, where each probe is individually packaged or dispensed; in other cases, the set is provided as a mixed collection of all probes in the set. Whether mixed or individually packaged, the set may contain components that contribute to stability and / or detection. These components may include one or more of buffers, primers, polymerases, nucleotides, thickeners, and fluorescence stabilizers to avoid photobleaching. The set may be dry (e.g., lyophilized) or provided in a suitable medium, with or without additional components. Suitable media may be aqueous (e.g., water-based) or organic. In combined implementations, the probes may be optimized or tailored.

[0104] As described above, the tunability of the implementation scheme facilitates multiplexing applications. This tunability can compensate for problems in detection, such as reduced emission and / or signal brightness of certain fluorophores, and compensate for variations within the sample, such as simultaneously detecting small and large amounts of targets within the sample. Therefore, probes within a probe group can have dye structures with different numbers of donor and / or acceptor fluorophores to compensate for variations in brightness and / or emission of probes within the group.

[0105] The dye structure of some embodiments can vary. In some cases, the dye structure of certain embodiments includes: a polymer backbone; one or more pendant donor fluorophores, each independently attached to a repeating unit of the polymer backbone; and one or more pendant acceptor fluorophores attached to a repeating unit of the polymer backbone, wherein the pendant donor fluorophores and pendant acceptor fluorophores are in an energy transfer relationship. Therefore, the dye structure of some embodiments includes one or more pendant donor fluorophores and one or more pendant acceptor fluorophores configured to be adjacent to one or more pendant donor fluorophores to receive energy, for example, both are attached to a common polymer backbone. In some embodiments, multiple pendant donor fluorophores are present and configured to be adjacent to pendant acceptor fluorophores for energy transfer, wherein in some cases, the number of pendant donor fluorophores ranges from 2 to 20, for example 2 to 15, for example 2 to 10. The term "pendant group" refers to a side-chain group attached to the backbone but not part of the backbone itself. In embodiments of the dye structure, the donor fluorophores are capable of transferring energy to the attached acceptor fluorophores. Therefore, the subject dye structure includes a connected acceptor signaling fluorophore adjacent to the donor fluorophore system for energy reception, i.e., adjacent to at least one connected donor fluorophore for energy reception. The specific configuration of the pendant groups can be determined and controlled by the arrangement of repeating units of the underlying polymer backbone (also referred to herein as a “modular scaffold”), to which the pendant groups are attached. The dye structure may include multiple water-soluble groups attached to the scaffold and / or any convenient location of the pendant groups to provide a water-soluble polymer dye. The polymer backbone, i.e., the modular scaffold, may consist of repeating units that form a polymer backbone with side-chain groups to which the side-chain groups can be attached. The repeating units can be arranged in various configurations to provide a dye structure with desired spectral characteristics. The distance and arrangement between the covalent attachment sites of the pendant donor fluorophore and acceptor fluorophore (when present) can be controlled to provide the 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-soluble 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 some cases, the WSG is a substituent attached to a pendant donor fluorophore or a pendant acceptor fluorophore. In some cases, each pendant donor fluorophore group is replaced by one or more WSGs. As used herein, the terms “water-soluble group,” “water-soluble group,” and WSG are used interchangeably and refer to a group or substituent that is adequately solubilized in an aqueous environment, such as under physiological conditions, and imparts improved water solubility to the attached molecule. WSGs can increase the solubility of a given dye structure in a solution primarily composed of aqueous solution compared to a control dye lacking WSGs. The water-soluble group can be any convenient hydrophilic group that is well solubilized in an aqueous environment. The solubility of the water-soluble dye structures of this disclosure under aqueous conditions makes them particularly suitable for a variety of bioassays. Various water-soluble polymer groups can be used as WSGs for the subject dyes. Any convenient water-soluble group (WSG) can be included in the dyes described herein to provide greater water solubility, for example as described above.

[0107] As described above, the dye structure of embodiments of the present invention includes 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 subject dye structure, 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 some cases, the ratio of donor fluorophores to acceptor fluorophores is 5:1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 6:1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 7:1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 8:1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 9:1. In some 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 have an energy transfer relationship. Therefore, in embodiments of the present invention, excitation of the donor can lead to energy transfer to and from the covalently attached acceptor signaling fluorophore. Energy transfer mechanisms between the donor chromophore and the attached acceptor signaling fluorophore include, for example, resonant energy transfer (e.g., Förster (or fluorescence) resonant energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. These energy transfer mechanisms can be over relatively short distances; that is, proximity of the chromophores in a light-collecting multi-chromophore system to each other and / or to the acceptor fluorophore provides effective energy transfer. Under conditions of effective energy transfer, when the wavelength of the incident light (“pump light”) is absorbed and transferred from the chromophore of the light-collecting chromophore, the emission of the luminescent acceptor fluorophore is more intense than when the luminescent acceptor fluorophore is directly excited by the pump light, thus amplifying the emission of the acceptor fluorophore. "Effective" energy transfer refers to the transfer of 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more) of the energy collected by the donor chromophore to the acceptor. "Amplification" means that when excited by energy transfer from a donor-donor light-collecting chromophore system, the signal from the acceptor fluorophore is 1.5 times or more than that of the acceptor fluorophore directly excited with incident light of equal intensity. The signal can be measured using any convenient method. In some cases, 1.5 times or more of signal refers to the intensity of the emitted light. In other cases, 1.5 times or more of signal refers to an increased signal-to-noise ratio. In certain embodiments of the dye structure, the emission of the acceptor fluorophore when excited by the chromophore is 1.5 times or more, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 8 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, or even more, compared to direct excitation of the acceptor fluorophore with incident light.

[0109] The dye structure of the embodiments of the present invention can have any convenient molecular weight (MW). In some cases, the MW of the dye structure can be expressed as the average molecular weight. In some cases, the average molecular weight of the dye structure is in the range of 500 to 500,000, for example 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000, or even in the range of 50,000 to 100,000 Daltons. In some cases, the molecular weight of the polymeric fluorescent dye is in the range of 5 to 75 kDa, 10 to 50 kDa, for example 15 to 45 kDa.

[0110] In this implementation, the fluorescence emission from the acceptor fluorophore provided by the subject dye structure is brighter than the emission that such fluorescent dyes can produce alone. The brightness of the emission from the dye structure can be 50 mM. -1 cm -1 or more, for example, 60 mM - 1 cm -1 or more, 70 mM -1 cm -1 or more, 80 mM -1 cm -1 or more, 90 mM -1 cm -1 or more, 100 mM -1 cm -1 or more, 150 mM -1 cm -1 or more, 200 mM -1 cm -1 or more, 250 mM -1 cm -1 or more, 300 mM -1 cm -1 Or higher, or even higher. In some cases, the emission brightness of the dye structure is at least 5 times higher than that of the directly excited acceptor fluorophore, for example, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 300 times, or even more.

[0111] In addition to the properties mentioned above, the dye structure of the embodiments of the present invention may also have one or more additional desired spectral characteristics, such as a specific maximum emission wavelength, extinction coefficient, quantum yield, etc.

[0112] Various emission spectra depend on a variety of factors, such as the selected comonomers, linking groups, substituents, and linked acceptor fluorophores contained in the dye structure.

[0113] In some embodiments, the dye structure may include fragments having the formulas described in one or more of PCT / US2019 / 024662 (published as WO2019 / 191482) and U.S. Patent Applications Nos. 18 / 201,940 and 18 / 120,554, the disclosures of which are incorporated herein by reference in their entirety. Further details regarding the WSG groups that may be found in the dye structures of embodiments of the invention can be found in PCT application serial number PCT / US2019 / 024662 (published as WO 2019 / 191482).

[0114] The general structure of a nucleic acid probe is shown in the following formula:

[0115] in: Each D is an independent pendant donor fluorophore (e.g., as described herein); Each A is an independent receptor chromophore (e.g., as described herein); l, m, and n are each an independent integer from 1 to 20; L stands for connector; R1 and R2 are independently selected from end groups, polymer segments, donor chromophore groups, water-soluble groups, linkers, and linkers with specific binding members; Nucleic acid is a nucleic acid having a sequence complementary to a target nucleic acid (e.g., as described herein); and Q is a quencher (e.g., as described herein).

[0116] In some cases of the preceding equation, l, m, and n are each independently 1 to 10, such as 2 to 20, 3 to 10, or 3 to 6. In some cases of the preceding equation, n is an integer from 2 to 20, such as 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In some implementations of the preceding equation, m is 1.

[0117] An exemplary (and non-limiting) representation of a nucleic acid probe according to certain implementations is shown below. Figure 2 According to Figure 2 The example provides a nucleic acid having a dye structure attached to its first end and a quencher attached to its second end. The dye structure comprises a non-conjugated polymer backbone formed of amino acids, such as a peptide structure. This example provides six donor fluorophores and one acceptor fluorophore according to the foregoing description.

[0118] method Aspects of the present invention include methods for assessing the presence of target nucleic acids in a sample. These methods include generating a reaction mixture comprising a sample and a nucleic acid probe. Such probes have been described in the foregoing disclosure and can be used to generate the reaction mixture. In some cases, the sample contains, is suspected of containing, may contain, or does not contain nucleic acids. In some cases, the sample comprises one or more copies of a target nucleic acid complementary to the nucleic acid probe. In some embodiments, the nucleic acid probe hybridizes with any copy of the target nucleic acid in the sample. Other embodiments monitor a signal emitted by a dye structure associated with the nucleic acid probe in the reaction mixture to determine the presence of the target nucleic acid in the sample.

[0119] In some cases, the method may include irradiating the reaction mixture with the excitation wavelength of one or more donor fluorophores. The specific wavelength, bandwidth, intensity, or other parameters may be selected by those skilled in the art and depend on which donor fluorophore is a component of the nucleic acid probe.

[0120] In some cases, the reaction mixture also contains a polymerase with 5'-3' nuclease activity, and monitoring of the reaction mixture includes performing a nucleic acid amplification reaction. Nucleic acid amplification can include isothermal amplification and polymerase chain reaction (PCR). In the amplification reaction described herein, a polymerase with 5'-3' nuclease activity polymerizes a new strand based on a target sequence or target region. When the polymerase encounters a hybridized nucleic acid probe, the nuclease activity hydrolyzes the nucleic acid that is part of the nucleic acid probe. This hydrolysis releases the dye structure from the quencher, thereby causing the dye structure to signal.

[0121] As is known to those skilled in the art, PCR includes providing a reaction mixture with a pair of amplification primers and a deoxyribonucleotide triphosphate, wherein the first amplification primer contains a sequence complementary to a region of one strand of the target nucleic acid and is capable of initiating 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 is capable of initiating the synthesis of the complementary strand thereto. In these embodiments, a polymerase exhibiting 5'-3' nuclease activity amplifies the target nucleic acid located between the amplification primers, and a nucleic acid probe is complementary to a region of the target nucleic acid located between the pair of amplification primers. PCR may further include cycling the reaction temperature of the sample to a first temperature suitable for denaturing the target nucleic acid, then cycling to a second temperature suitable for hybridizing the pair of amplification primers and the nucleic acid probe with the target nucleic acid, and then cycling to a third temperature suitable for the polymerase to amplify the target nucleic acid. This cycling process may be iterated any number of times sufficient to cause the dye structure to signal. Some embodiments cycle the reaction temperature 4-35 times, including at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, or at least 25 times.

[0122] The reaction temperature for PCR can be appropriately selected based on the specific reaction type, the reaction mixture, and / or the components contained in the reaction mixture. In PCR, the first denaturation temperature can be equal to or higher than the temperature at which any double-stranded nucleic acid is separated. This first temperature can be at or near 100°C, such as 90°C, 92°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C. However, lower temperatures may be used in certain circumstances that will be understood by those skilled in the art. The second temperature suitable for primer hybridization or annealing is typically the melting temperature (T0) of the primers and / or probes. mThe temperature may be 55°C or below. Typically, this range is between 55°C and 72°C, but some implementations may use lower temperatures. Furthermore, this second temperature can be adjusted between iterations, such as step-up PCR (increasing temperature with each iteration), touchdown PCR (decreasing temperature with each iteration), and / or any other cycling strategy to improve sensitivity and / or detection. Finally, the suitable third temperature for extension depends on the specific enzyme to ensure continuity and / or efficiency during the extension or polymerization of the new nucleic acid strand. This temperature is typically around 72°C, but can vary depending on the enzyme. Therefore, the third temperature can be selected from about 65°C to about 80°C, including 70°C, 72°C, 75°C, and / or 78°C.

[0123] In some cases, probes as described herein are implemented in real-time PCR (including quantitative PCR (qPCR)). While real-time PCR and qPCR can use non-specific dyes to quantify amplification, these methods can measure non-specific amplification and therefore have limited accuracy. As described herein, probes improve the accuracy of qPCR by detecting specific amplicons. Such probes hybridize or anneal to the target sequence (i.e., the probe's complementary sequence) at the same stage as hybridization with primers. During the extension phase, a polymerase with 5'–3' exonuclease activity hydrolyzes or digests the nucleic acid portion of the probe. Hydrolysis releases the scaffolded chromophore, or dye structure, from the probe and allows the dye structure to diffuse within the reaction mixture. This release disrupts the energy transfer relationship between the acceptor fluorophore and the quencher on the dye structure, thereby allowing the measurement or imaging of the fluorescence signal. In a qPCR reaction, imaging or detection can be performed continuously throughout the reaction or at a single time point during temperature cycling. For example, imaging can be performed after extension but before denaturation. However, imaging or detection can also be performed during denaturation and / or hybridization. Detailed information regarding the application of real-time PCR and / or qPCR in embodiments of this invention is further described in U.S. Patent Nos. 11,001,877, 9,944,978, 8,697,362, 8,137,616, 8,017,380, and 7,101,663; and published U.S. Patent Application Publications Nos. 2020 / 0370097, 2012 / 0088691, 2011 / 0300544, and 2005 / 0053950, the disclosures of which are incorporated herein by reference in their entirety.

[0124] Isothermal amplification can be performed by various methods known in the art, including sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), and rolling circle amplification (RCA). In addition to polymerases with 5'-3' nuclease activity, such reaction mixtures may also contain one or more primers, ligases, recombinases, deoxyribonucleotides, ribonucleotides, one or more adaptors, and various other components known to those skilled in the art. The isothermal amplification reaction temperature can depend on the reaction type, enzyme, and / or any other factors. In some embodiments, the reaction occurs between approximately 30°C and 45°C, such as 32°C, 35°C, 37°C, 40°C, and 42°C. Some embodiments use 37°C as the reaction temperature for the isothermal amplification reaction.

[0125] Nucleic acid amplification, such as that described herein, may further include components for auxiliary reactions, including buffers, solvents, and / or any other components. Such buffers may include HEPES, PBS, SSC, Tris, any other buffer for a specific enzyme, and combinations thereof. Solvents may also be used to improve specificity, reaction efficiency, and / or any other properties to facilitate such reactions. Dimethyl sulfoxide (DMSO) is a commonly used solvent in many reactions. As those skilled in the art will appreciate, the reaction mixture may contain other components.

[0126] Furthermore, the methods described herein can be used in multiplexing reactions with the second nucleic acid probe described herein. When using the second probe, the relevant nucleic acid is complementary to a second target sequence that is different from the first target sequence. During the amplification reaction, the second probe can hybridize with a pair of amplification primers of the first nucleic acid probe, or with a region between a pair of amplification primers of the second probe. In some multiplexing embodiments, the number of different target nucleic acids measured simultaneously varies. In some cases, the number of different target nucleic acids measured simultaneously is two or more, such as five or more, including ten or more, such as 25 or more, 50 or more, 75 or more, etc. In some cases, the number can be 1000 or less, such as 500 or less, including 250 or less, such as 200 or less, 150 or less, including 100 or less. The number of target nucleic acids to be measured can 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.

[0127] In some embodiments, the sample can be any substance that actually contains or may contain nucleic acids. Such samples can be biological or environmental samples. Biological samples include animals, plants, fungi, bacteria, viruses, and / or archaea. The sample can be a whole organism, a single cell, or any part thereof. Certain samples can be derived from a tissue, an organ, a biological product (such as blood), or any combination thereof. In some embodiments, biological samples are collected in the form of saliva, feces, urine, sweat, sebum, mucus, semen, breast milk, blood, and / or any other biological product. Blood samples can be collected in the form of whole blood, peripheral whole blood with lysed erythrocytes before cell separation, 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 tissues (such as liver, kidney, brain, skin, breast, fat, bone, bone marrow, etc.) or abnormal tissues (such as growths, cancer, tumors, lesions, etc.).

[0128] Environmental samples can be used to identify the presence of certain species or organisms without direct eyewitness testimony or other evidence of their presence. Such samples can include those collected from air, water, sewers, the ground, toilets, sinks, door handles, and / or any other non-biological location or material.

[0129] Fluorometers can be used to measure fluorescence in samples. In some cases, excitation radiation from an excitation source having a first wavelength passes through an excitation optics. The excitation optics induce excitation radiation to excite fluorophores within the reaction mixture. In response, a fluorescently labeled target in the sample emits radiation at a wavelength different from the excitation wavelength. A collection optics then collects the emission from the reaction mixture. The apparatus may include a temperature controller to maintain the sample at a specific temperature during scanning. In some cases, a multi-axis translation stage moves a microtiter plate holding multiple samples to position different wells to be exposed. The multi-axis translation stage, temperature controller, autofocus function, and electronics associated with imaging and data collection can be programmed by a suitably programmed digital computer. The computer can also convert the data collected during the measurement into another format for presentation.

[0130] In some embodiments, the method for assessing the presence of the target nucleic acid in a sample further includes detecting fluorescence in a flow cytometer. In some embodiments, the method for assessing the presence of the target nucleic acid in a sample further includes imaging the sample in contact with the labeled composition using fluorescence microscopy. 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. Microscopic methods of interest that can be used in the subject method include laser scanning confocal microscopy.

[0131] Since some methods for detecting target nucleic acids utilize nucleic acid amplification technology, fluorescence can be detected using a real-time thermal cycler or a thermal cycler with an additional excitation and detection array to monitor fluorescence emission from the reaction mixture.

[0132] Hybrid products Some embodiments of this disclosure include products generated using the nucleic acid probes disclosed herein. Such products may include probes that hybridize with a target nucleic acid. These products may include a first nucleic acid and a second nucleic acid, each containing multiple nucleotides forming a chain from the 5' end to the 3' end. Furthermore, at least a portion of the first nucleic acid and the second nucleic acid are complementary. The first nucleic acid may be a nucleic acid probe as described herein and includes the dye structure and quencher described herein. The first nucleic acid may include a second quencher, minor groove binding agent, water-soluble groups, etc., as described above.

[0133] The second nucleic acid may include a target nucleic acid. Such nucleic acid may be DNA, RNA, or other forms of nucleic acid (e.g., as described herein). Such targets may be nuclear DNA and / or genomic DNA, messenger RNA (mRNA), precursor mRNA, ribosomal RNA, transfer RNA, amplicon, and / or any other nucleic acid that may serve as a target and / or of interest.

[0134] system The invention also includes systems for practicing the subject methods and compositions. Sample analysis systems may include a sample-containing chamber as described herein. Systems of this embodiment may include an illumination source oriented to illuminate the sample and a detector, oriented such that light emitted from the sample illuminates the detector. The sample may contain nucleic acid probes as described, and may contain or be suspected of containing target nucleic acids. Further embodiments of the system may include heating elements for changing the temperature of the sample. Such heating elements may include a water bath or a hot block, and may use thermoelectric effects to change the temperature of the sample.

[0135] Further implementations may include a user interface that allows selection of temperature, irradiation wavelength, detection wavelength, and temperature cycling 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 system conforming to the description herein.

[0136] In some aspects, the system may further include a computer-based system configured to detect the presence of a fluorescence signal. "Computer-based system" refers to hardware devices, software devices, and data storage devices used to analyze the information of this invention. The minimum hardware of the computer-based system of this invention includes a central processing unit (CPU), input devices, output devices, and data storage devices. Those skilled in the art will readily recognize that any currently available computer-based system is applicable to the subject system. The data storage device may include any article of writing that includes the information described above, or a storage accessor that can access such article of writing.

[0137] "Recording" data, programming, or other information on a computer-readable medium refers to the process of storing information using any such methods known in the art. Any convenient data storage structure can be chosen based on the means used to access the stored information. A variety of data processing programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0138] "Processor" refers to any combination of hardware and / or software that performs its required functions. For example, any processor described herein can be a programmable digital microprocessor, such as those available in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). Where the processor is programmable, appropriate programming can be transferred to the processor from a remote location or pre-stored in a computer program product (such as a portable or stationary computer-readable storage medium, whether based on a magnetic, optical, or solid-state device). For example, a magnetic medium or optical disc can carry the program and can be read by a suitable reader that communicates with each processor located at its corresponding site.

[0139] In addition to sensor devices and signal processing modules, as described above, the system of the present invention may include many additional components, such as data output devices, such as displays and / or speakers, data input devices (e.g., interface terminals, keyboards, etc.), fluid processing components, power supplies, etc.

[0140] Other systems may also be used to practice the subject method. In some aspects, the system may be a fluorometer or microscope loaded with a sample having any of the embodiments discussed herein. The fluorometer or microscope may include a light source configured to direct light to a measurement area or field of view of the sample in a flow channel. The fluorometer or microscope may also include a detector configured to receive a signal from the measurement area or field of view of the flow channel, wherein the signal is provided by the fluorescent composition.

[0141] Reagent test kit The invention further includes kits for practicing the subject methods and compositions. The compositions of the invention, such as one or more probes (e.g., as described above), may be included in the kit as reagents or as starting materials, or provided for use in, for example, the methods described above. The kit may comprise probes (e.g., as described above) and a container. Any convenient container may be used, such as tubes, bottles, or wells in multi-well strips or plates, boxes, bags, insulated containers, etc. The subject kit may also include, as needed, one or more components selected from probes for a given target sequence, support-binding probes, cells, supports, biocompatible aqueous elution buffers, controls (positive and / or negative), etc., and instructions for use. A given kit may include reagents suitable for detecting a single target nucleic acid, or multiple reagents suitable for detecting two or more different target nucleic acids, for example, wherein the given kit is configured for multiplexing detection applications.

[0142] In some implementations, the kit is used to assess the location of a target sequence in a sample, such as through in situ hybridization. Therefore, in some cases, the kit contains one or more components suitable for permeabilizing or lysing cells. One or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in multi-well strips or plates).

[0143] In some cases, the kit also includes reagents for nucleic acid amplification. Reagents of interest include, but are not limited to: buffers for reconstruction and dilution, buffers for contacting the sample with the nucleic acid probe, wash buffers, control reagents, enzymes (e.g., polymerases, polymerases with 5'-3' nuclease activity, recombinases, ligases, etc.), nuclease-free water, nucleoside triphosphates, deoxynucleoside triphosphates, one or more primers, adapters, and combinations thereof. Additionally, the kit may contain cell permeation reagents such as methanol, acetone, or detergents (e.g., Triton, NP-40, saponins, Tween 20, digitalis saponins, leucoperm, or any combination thereof or buffers).

[0144] The kit compositions may be provided as liquid compositions, such as any suitable buffer. Alternatively, the kit compositions may be provided as dry compositions (e.g., lyophilized), and the kit may optionally contain one or more buffers for reconstitution of the dry compositions. In some aspects, the kit may contain aliquots of the compositions provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0145] Furthermore, one or more components may be combined into a single container, such as a glass or plastic vial, tube, or bottle. In some cases, the kit may further include a container (e.g., box, bag, insulated container, bottle, tube, etc.) containing all components (and their individual containers). The kit may further include packaging that is separate from or attached to the kit container and that is printed with information about the kit, components, and / or instructions for use.

[0146] In addition to the components described above, the subject kit may further include instructions for practicing the subject methodology. These instructions may exist in various forms within the subject kit, with one or more of them present in the kit itself. One form of these instructions is as printed information on a suitable medium or substrate (e.g., one or more sheets of paper with the information printed on them), as packaging material for the kit, as inserts in the packaging, etc. Another means is as computer-readable media, such as floppy disks, CDs, DVDs, portable flash drives, etc. Yet another means is as a website address, which can be used via the Internet to access information at a removed site. Any convenient means may be present in the kit.

[0147] use The scaffold-type chromophores for nucleic acid detection described herein, as well as the probes, compositions, methods, and systems incorporating such chromophores, can be used in a variety of applications, including diagnostic and research applications where labeling, detection, and / or analysis of a target of interest is required. Such applications include methods such as cell counting, 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 can be used to analyze any of a variety of samples, including but not limited to biofluids, cell culture samples, and tissue samples. In some respects, the compositions, systems, and methods described herein can be used in methods where fluorescent labeling is used to detect analytes in a sample (if present), such as fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, etc. In some cases, the compositions and methods are used in applications where the presence of a target analyte in a sample is of interest to be evaluated.

[0148] In some cases, the methods and compositions are used in any form of assay in which there is interest in detecting and / or analyzing targets 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 some cases, the methods and compositions are used in any application in which there is interest in fluorescently labeling target molecules. The subject composition is suitable for any convenient application in which nucleic acid probes are available.

[0149] The following examples are provided for illustrative purposes and not for limitation.

[0150] Example Example 1 - Real-time PCR Various nucleic acid probes will be incorporated into real-time PCR (rtPCR). rtPCR can be quantitative and is known as quantitative PCR (qPCR). In rtPCR, a reaction mixture is generated according to a standard PCR reaction, and probes that can identify specific sequences that may be present in the sample are added. The reaction mixture will contain sufficient amounts of buffer, nucleotides (e.g., deoxyribonucleotides and / or ribonucleotides), polymerase (e.g., Taq polymerase), primers, template, and probes, such as... Figure 2 The probe is shown. The reaction mixture may contain the following components and amounts:

[0151] Once the reaction mixture is produced, it will be incubated in a suitable thermal cycler, where a real-time thermal cycler is typically equipped with a light source and detector to allow for the excitation of fluorophores and the detection of any fluorophore emission. The thermal cycler will perform temperature cycling to allow for denaturation, primer and probe annealing, and primer extension. The cycling reaction can follow this procedure: (1) Initial denaturation at 95℃ for 3 minutes (2) Denaturation at 95℃ for 1 minute (3) Anneal at 55℃ for 2 minutes (4) Extend to 72℃ for 3 minutes (5) Imaging / Detection (6) Repeat steps 2-5 for 10-45 cycles. During elongation, the exonuclease activity of the polymerase hydrolyzes nucleic acids, releasing the donor and acceptor fluorophores. This release allows the fluorophores to diffuse from the quencher. This diffusion eliminates the energy transfer relationship between the fluorophore and the quencher, thus enabling light emission.

[0152] The reaction will be monitored by appropriate excitation and emission maxima. Using the probe shown in Figure 3, appropriate excitation wavelengths will include the excitation maxima of the six donor fluorophores. Detection wavelengths will include the emission maxima of the acceptor fluorophore. Monitoring can be performed continuously throughout the reaction procedure or in separate detection or imaging steps.

[0153] Notwithstanding the appended claims, this disclosure is further defined by the following: Item 1. A probe for nucleic acid detection, said probe comprising: A nucleic acid complementary to a target sequence, wherein the nucleic acid comprises multiple nucleotides forming a chain from the 5' end to the 3' end; A dye structure attached to the first end of the nucleic acid, wherein the dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the nucleic acid, wherein the quencher and the one or more receptor fluorophores have an energy transfer relationship.

[0154] Item 2. The probe according to Item 1, wherein the nucleic acid comprises one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA).

[0155] Item 3. The probe according to Item 1 or 2, wherein the base associated with each nucleotide of the plurality of nucleotides is selected from standard bases, modified bases and base analogs.

[0156] Item 4. The probe according to Item 3, wherein at least one of the plurality of nucleotides is a nucleotide analog.

[0157] Item 5. The probe according to any one of items 1-4 further comprises a small groove adhesive (MGB) linked to the nucleic acid.

[0158] Item 6. The probe according to Item 3, wherein the MGB is attached to the 3' end of the nucleic acid.

[0159] Item 7. The probe according to any of the preceding items, wherein the plurality of nucleotides comprises 10-50 nucleotides.

[0160] Item 8. The probe according to Item 7, wherein the plurality of nucleotides comprises 18-30 nucleotides.

[0161] Item 9. The probe according to any of the preceding items, wherein the one or more excitable donor fluorophores have an excitation maximum ranging from 300 to 700 nm.

[0162] Item 10. The probe according to Item 9, 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.

[0163] Item 11. The probe according to Item 9 or 10, wherein the one or more excitable donor fluorophores have emission maximum values ​​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.

[0164] Item 12. The probe according to any of the preceding items, wherein the one or more excitable donor fluorophores are selected from bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0165] Item 13. The probe according to any of the preceding items, wherein one or more excitable donor fluorophores are bonded to a water-soluble group.

[0166] Item 14. The probe according to Item 13, wherein the water-soluble group includes a nonionic water-soluble group.

[0167] Item 15. The probe according to Item 14, wherein the nonionic water-soluble group comprises polyalkylene glycol.

[0168] Item 16. The probe according to Item 15, wherein the polyalkylene glycol comprises polyethylene glycol.

[0169] Item 17. The probe according to any of the preceding items, wherein the maximum excitation value of the one or more acceptor fluorophores ranges from 450 to 750 nm.

[0170] Item 18. The probe according to Item 17, wherein the one or more acceptor fluorophores have an excitation maximum ranging from 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.

[0171] Item 19. The probe according to any one of items 1 to 18, wherein the maximum emission range of the one or more acceptor fluorophores is 500 to 875 nm.

[0172] Item 20. The probe according to Item 19, wherein the one or more acceptor fluorophores have emission maximum values ​​ranging from 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0173] Item 21. The probe according to any of the preceding items, wherein the one or more acceptor fluorophores are selected from coumarin dyes, cyanine dyes, rhodamine dyes, xanthan dyes, polyacetylenes, dipyrrole methyl boron difluoride, naphthalimide, thiazide dyes, and acridine dyes.

[0174] Item 22. The probe according to any of the preceding items, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

[0175] Item 23. The probe according to Item 22, wherein the number of donor fluorophores ranges from 2 to 20.

[0176] Item 24. The probe according to any of the preceding probes, wherein the dye structure comprises a single acceptor fluorophore.

[0177] Item 25. The probe according to any of the preceding items, wherein the non-conjugated polymer backbone comprises at least one of peptide, carbohydrate, hydrocarbon, polyethylene glycol (PEG) polymer, lipid, peptide-like substance, and polynucleotide.

[0178] Item 26. The probe according to Item 25, wherein the non-conjugated polymer backbone comprises a peptide.

[0179] Item 27. The probe according to Item 26, wherein the peptide comprises 5 to 100 amino acid residues.

[0180] Item 28. The probe according to any of the preceding items, wherein the quencher absorbs light at the emission maximum of the one or more acceptor fluorophores.

[0181] Item 29. The probe according to any of the preceding items, wherein the quencher absorbs light in the range of 450 to 875 nm.

[0182] Item 30. The probe according to Item 29, wherein the quencher absorbs light in the range of 440 to 485 nm, 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0183] Item 31. The probe according to Item 29 or 30, wherein the quencher is selected from Black HoleQuencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher.

[0184] Item 32. The probe according to Item 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, QXL 490, QXL 570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl) or tetramethylrhodamine (TAMRA).

[0185] Item 33. The probe according to any of the preceding items, wherein the dye structure is attached to the 5' end of the nucleic acid and the quencher is attached to the 3' end of the nucleic acid.

[0186] Item 34. The probe according to any one of items 1 to 32, wherein the dye structure is attached to the 3' end of the nucleic acid and the quencher is attached to the 5' end of the nucleic acid.

[0187] Item 35. The probe according to Item 33 or 34 further includes a second quencher linked to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid.

[0188] Item 36. The probe according to any one of items 33 to 35, wherein the second quencher is linked to a nucleotide located at 9 or 10 nucleotides away from the 5' end of the nucleic acid.

[0189] Item 37. The probe according to Item 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, QXL 490, QXL 570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphorus amide quencher, azo quencher, or anthraquinone quencher.

[0190] Item 38. A probe set for nucleic acid detection, said set comprising: A first nucleic acid probe, the first nucleic acid probe comprising: A first nucleic acid complementary to a first target sequence, wherein the first nucleic acid comprises a plurality of nucleotides forming a chain from the 5' end to the 3' end; A first dye structure is attached to a first end of the first nucleic acid, wherein the first dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the first nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; and A second nucleic acid probe, the second nucleic acid probe comprising: A second nucleic acid complementary to a second target sequence, wherein the second nucleic acid comprises a plurality of nucleotides forming a chain from the 5' end to the 3' end; A second dye structure is attached to the first end of the second nucleic acid, wherein the second dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the second nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; Wherein the first target sequence is different from the second target sequence; and The first dye structure has a different emission maximum value than the second dye structure.

[0191] Item 39. The group according to Item 38, wherein the first nucleic acid and the second nucleic acid consist of one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA).

[0192] Item 40. The group according to Item 38 or 39, wherein the nucleoside associated with each of the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid is selected from standard nucleosides, modified nucleosides and nucleoside analogs.

[0193] Item 41. The group according to Item 40, wherein at least one nucleoside of the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid is a nucleoside analog.

[0194] Item 42. The group according to any one of items 38 to 41, wherein one or more of the first nucleic acid probe and the second nucleic acid probe further comprises a small groove adhesive (MGB) connected thereto.

[0195] Item 43. The group according to Item 42, wherein the MGB is connected to the 3' end of one or more of the first nucleic acid probe and the second nucleic acid probe.

[0196] Item 44. The group according to any one of items 38 to 43, wherein the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid comprises 10-50 nucleotides.

[0197] Item 45. The group according to Item 44, wherein each of the first nucleic acid and the second nucleic acid comprises 18-30 nucleotides.

[0198] Item 46. The group according to any one of items 38 to 45, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum ranging from 300 to 700 nm.

[0199] Item 47. The group according to any one of items 38 to 46, 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 maximum values.

[0200] Item 48. The group according to Item 46 or 47, 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.

[0201] Item 49. The group according to any one of items 46 to 48, wherein the one or more excitable donor fluorophores have emission maximum values ​​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.

[0202] Item 50. The group according to any one of items 38 to 49, wherein the one or more excitable donor fluorophores are selected from bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0203] Item 51. The group according to any one of items 38 to 50, wherein the one or more excitable donor fluorophores are bonded to a water-soluble group.

[0204] Item 52. The group according to Item 51, wherein the water-soluble group includes nonionic water-soluble groups.

[0205] Item 53. The group according to Item 52, wherein the nonionic water-soluble group comprises polyalkylene glycols.

[0206] Item 54. The group according to Item 53, wherein the polyalkylene glycol comprises polyethylene glycol.

[0207] Item 55. The group according to any one of items 38 to 54, wherein the maximum excitation range of the one or more acceptor fluorophores is 450 to 750 nm.

[0208] Item 56. The group according to any one of items 38 to 55, wherein the one or more receptor fluorophores associated with the first nucleic acid probe and the one or more receptor fluorophores associated with the second nucleic acid probe have different emission maximum values.

[0209] Item 57. The group according to Item 56, wherein the one or more acceptor fluorophores have an excitation maximum ranging from 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.

[0210] Item 58. The group according to any one of items 38 to 57, wherein the one or more acceptor fluorophores are selected from coumarin dyes, cyanine dyes, rhodamine dyes, xanthan dyes, polyacetylenes, boron difluoride dipyrrole, naphthalimide, thiazide dyes, and acridine dyes.

[0211] Item 59. The group according to any one of items 38 to 58, wherein in each of the first nucleic acid probe and the second nucleic acid probe, the number of donor fluorophores exceeds the number of recipient fluorophores.

[0212] Item 60. The group according to Item 59, wherein the number of donor fluorophores ranges from 2 to 20.

[0213] Item 61. The group according to Item 60, wherein the first dye structure comprises a different amount of donor fluorophore than the second dye structure.

[0214] Item 62. The group according to any one of items 38 to 61, wherein the first dye structure and the second dye structure each comprise a single acceptor fluorophore.

[0215] Item 63. The group according to any one of items 38 to 61, wherein the first dye structure comprises an amount of acceptor fluorophore different from that of the second dye structure.

[0216] Item 64. The group according to any one of items 38 to 63, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptide-like substances, and polynucleotides.

[0217] Item 65. The group according to Item 64, wherein each of the non-conjugated polymer backbones comprises a peptide.

[0218] Item 66. The group described in Item 65, wherein each peptide contains 5 to 100 amino acid residues.

[0219] Item 67. The group according to any one of items 38 to 66, wherein the quencher absorbs light at the emission maximum of the one or more acceptor fluorophores.

[0220] Item 68. The group according to any one of items 38 to 67, wherein the quencher absorbs light in the range of 450 to 875 nm.

[0221] Item 69. The group according to Item 68, wherein the quencher absorbs light in the range of 440 to 485 nm, 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0222] Item 70. The group according to any one of items 38 to 69, wherein the quencher is selected from Black HoleQuencher (BHQ), QSY quencher, Iowa Black quencher or QXL quencher.

[0223] Item 71. The group according to any one of items 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, QXL 490, QXL570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl) or tetramethylrhodamine (TAMRA).

[0224] Item 72. The group according to any one of items 38 to 71, wherein the first dye structure and the second dye structure are attached to the 5' end of the first nucleic acid and the second nucleic acid, and the quencher is attached to the 3' end of the first nucleic acid and the second nucleic acid.

[0225] Item 73. The group according to any one of items 38 to 72, wherein the first dye structure and the second dye structure are attached to the 3' end of the first nucleic acid and the second nucleic acid, and the quencher is attached to the 5' end of the first nucleic acid and the second nucleic acid.

[0226] Item 74. The group according to Item 79 or 73, wherein one or both of the first nucleic acid probe and the second nucleic acid probe further comprises a second quencher linked to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid.

[0227] Item 75. The group according to any one of items 72 to 74, wherein the second quencher is linked to a nucleotide located at 9 or 10 nucleotides away from the 5' end of the nucleic acid.

[0228] Item 76. The group according to Item 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, QXL 490, QXL 570, QXL670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphorus amide quencher, azo quencher, or anthraquinone quencher.

[0229] Item 77. A kit for nucleic acid detection, said kit comprising: A first nucleic acid probe, the first nucleic acid probe comprising: A first nucleic acid complementary to a first target sequence, wherein the first nucleic acid comprises a plurality of nucleotides forming a chain from the 5' end to the 3' end; A first dye structure is attached to a first end of the first nucleic acid, wherein the first dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the first nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship.

[0230] Item 78. The kit according to Item 77 further comprises: A second nucleic acid probe, the second nucleic acid probe comprising: A second nucleic acid complementary to a second target sequence, wherein the second nucleic acid comprises a plurality of nucleotides forming a chain from the 5' end to the 3' end; A second dye structure is attached to the first end of the second nucleic acid, wherein the second dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the second nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; Wherein the first target sequence is different from the second target sequence; and The first dye structure has a different emission maximum value than the second dye structure.

[0231] Item 79. The kit according to Item 77 or 78, wherein the first nucleic acid and the second nucleic acid consist of one or more of the following: deoxyribonucleic acid (DNA) and locked nucleic acid (LNA).

[0232] Item 80. The kit according to any one of items 77 to 79, wherein the base associated with each of the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid is selected from standard bases, modified bases and base analogs.

[0233] Item 81. The kit according to Item 80, wherein at least one nucleotide of the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid is a nucleotide analog.

[0234] Item 82. A kit according to any one of items 77 to 81, wherein at least one nucleic acid probe further comprises a minor groove binding agent (MGB).

[0235] Item 83. The kit according to Item 82, wherein the MGB is attached to the 3' end of the nucleic acid probe.

[0236] Item 84. The kit according to any one of items 77 to 83, wherein the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid comprises 10-50 nucleotides.

[0237] Item 85. The kit according to Item 84, wherein each of the first nucleic acid and the second nucleic acid comprises 18-30 nucleotides.

[0238] Item 86. The kit according to any one of items 77 to 85, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum ranging from 300 to 700 nm.

[0239] Item 87. The kit according to any one of items 77 to 86, 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 maximum values.

[0240] Item 88. The kit according to Item 86 or 87, wherein the one or more excitable donor fluorophores have emission maximum values ​​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.

[0241] Item 89. The kit according to Item 88, wherein the one or more excitable donor fluorophores have emission maximum values ​​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.

[0242] Item 90. The kit according to any one of items 77 to 89, wherein the one or more excitable donor fluorophores are selected from bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0243] Item 91. The kit according to any one of items 77 to 90, wherein the one or more excitable donor fluorophores are bonded to a water-soluble group.

[0244] Item 92. The kit according to Item 91, wherein the water-soluble group comprises a nonionic water-soluble group.

[0245] Item 93. The kit according to Item 92, wherein the nonionic water-soluble group comprises polyalkylene glycol.

[0246] Item 94. The kit according to Item 93, wherein the polyalkylene glycol comprises polyethylene glycol.

[0247] Item 95. The kit according to any one of items 77 to 94, wherein the maximum excitation range of the one or more acceptor fluorophores is 450 to 750 nm.

[0248] Item 96. The kit according to any one of items 77 to 95, wherein the one or more receptor fluorophores associated with the first nucleic acid probe and the one or more receptor fluorophores associated with the second nucleic acid probe have different emission maximum values.

[0249] Item 97. The kit according to Item 95 or 96, wherein the one or more receptor 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.

[0250] Item 98. The kit according to any one of items 77 to 97, wherein the maximum emission range of the one or more acceptor fluorophores is 500 to 875 nm.

[0251] Item 99. The kit according to Item 98, wherein the one or more receptor fluorophores have emission maximum values ​​in the range of 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0252] Item 100. The kit according to any one of items 77 to 99, wherein the one or more receptor fluorophores are selected from coumarin dyes, cyanine dyes, rhodamine dyes, xanthan dyes, polyacetylenes, dipyrrole methyl boron difluoride, naphthalimide, thiazide dyes, and acridine dyes.

[0253] Item 101. The kit according to any one of items 77 to 100, wherein in each of the first nucleic acid probe and the second nucleic acid probe, the number of donor fluorophores exceeds the number of recipient fluorophores.

[0254] Item 102. The kit according to Item 101, wherein the number of donor fluorophores ranges from 2 to 20.

[0255] Item 103. The kit according to any one of items 77 to 102, wherein the first dye structure and the second dye structure each contain a single receptor fluorophore.

[0256] Item 104. The kit according to any one of items 77 to 103, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptide-like substances, and polynucleotides.

[0257] Item 105. The kit according to Item 104, wherein each of the non-conjugated polymer backbones comprises a peptide.

[0258] Item 106. The kit according to Item 1105, wherein the peptide comprises 5 to 100 amino acid residues.

[0259] Item 107. The kit according to any one of items 77 to 106, wherein the quencher absorbs light at the emission maximum of the one or more acceptor fluorophores.

[0260] Item 108. The kit according to any one of items 77 to 107, wherein the quencher absorbs light in the range of 450 to 875 nm.

[0261] Item 109. The kit according to Item 108, wherein the quencher absorbs light in the range of 440 to 485 nm, 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0262] Item 110. The kit according to any one of items 77 to 109, wherein the quencher is selected from Black Hole Quencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher.

[0263] Item 111. The kit according to any one of items 77 to 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, QXL 490, QXL 570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl) or tetramethylrhodamine (TAMRA).

[0264] Item 112. The kit according to any one of items 77 to 111, wherein the first dye structure and the second dye structure are linked to the 5' end of the first nucleic acid and the second nucleic acid, and the quencher is linked to the 3' end of the first nucleic acid and the second nucleic acid.

[0265] Item 113. The kit according to any one of items 77 to 111, wherein the first dye structure and the second dye structure are linked to the 3' end of the first nucleic acid and the second nucleic acid, and the quencher is linked to the 5' end of the first nucleic acid and the second nucleic acid.

[0266] Item 114. The kit according to Item 112 or 113, wherein one or both of the first nucleic acid probe and the second nucleic acid probe further include a second quencher linked to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid.

[0267] Item 115. The kit according to any one of items 112 to 114, wherein the second quencher is linked to a nucleotide located at 9 or 10 nucleotides away from the 5' end of the nucleic acid.

[0268] Item 116. The kit according to Item 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, QXL 490, QXL570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphorus amide quencher, azo quencher, or anthraquinone quencher.

[0269] Item 117. The kit according to any one of items 77 to 116 further comprises one or more of the following: buffer, water, deoxynucleoside triphosphate, nucleoside triphosphate, polymerase, 5'-3' nuclease and polymerase having 5'-3' nuclease activity.

[0270] Item 118. A method for determining the presence of a target nucleic acid in a sample, the method comprising: (a) Generating a reaction mixture, the reaction mixture comprising: The sample; and Nucleic acid probe, the nucleic acid probe comprising: A nucleic acid complementary to the target nucleic acid, wherein the nucleic acid comprises a plurality of nucleotides forming a chain from the 5' end to the 3' end. A dye structure attached to the first end of the nucleic acid, wherein the dye structure comprises: Non-conjugated polymer backbone, One or more donor fluorophores connected to the non-conjugated polymer backbone, and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship, and A quencher attached to the second end of the nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; and The nucleic acid probe hybridizes with any copy of the target nucleic acid in the sample; and (b) Monitoring the reaction mixture in response to a signal emitted from the dye structure to determine the presence of the target nucleic acid in the sample.

[0271] Item 119. The method according to Item 118 further includes irradiating the reaction mixture with the excitation wavelength of the one or more donor fluorophores.

[0272] Item 120. The method according to any one of Items 118 to 119, wherein the reaction mixture further comprises a polymerase having 5'-3' nuclease activity, and wherein monitoring the sample further comprises performing a polymerase chain reaction (PCR) on the reaction mixture.

[0273] Item 121. The method according to Item 120, wherein the PCR comprises: The reaction mixture is provided with amplification primer pairs and deoxyribonucleoside triphosphates; The first amplification primer contains a sequence complementary to a region of one strand of the target nucleic acid and is capable of initiating the synthesis of the complementary strand. The second primer contains a sequence complementary to a region of the second strand of the target nucleic acid and is capable of initiating the synthesis of the complementary strand. The polymerase exhibiting 5'-3' nuclease activity amplifies the target nucleic acid located between the amplification primers, and The nucleic acid probe is complementary to the region of the target nucleic acid located between the amplification primers; and The reaction temperature of the sample is cycled to a first temperature suitable for denaturing the target nucleic acid, then cycled to a second temperature suitable for allowing the amplification primer pair and the nucleic acid probe to hybridize with the target nucleic acid, and then cycled to a third temperature suitable for the polymerase to amplify the target nucleic acid.

[0274] Item 122. The method according to Item 121, wherein monitoring the sample further includes iterating the reaction temperature in the cycle for at least four iterations.

[0275] Item 123. The method according to any one of Items 118 to 119, wherein the reaction mixture further comprises a polymerase having 5'-3' nuclease activity, and wherein monitoring the sample further comprises performing an isothermal amplification reaction.

[0276] Item 124. The method according to Item 123, wherein the isothermal amplification reaction comprises employing one or more of the following: sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), and rolling circle amplification (RCA).

[0277] Item 125. The method according to any one of items 118 to 124, wherein: The reaction mixture also contains a second nucleic acid probe. The second nucleic acid probe comprises: A second nucleic acid complementary to a second target nucleic acid, wherein the second nucleic acid comprises multiple nucleotides forming a chain from the 5' end to the 3' end. A second dye structure is attached to the first end of the second nucleic acid, wherein the second dye structure comprises: Non-conjugated polymer backbone, One or more donor fluorophores connected to the non-conjugated polymer backbone, and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship, and A quencher attached to the second end of the nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; and The sample contained or was suspected of containing a second target nucleic acid; The target sequence thereon is different from the second target sequence; and The dye structure thereon has a different emission maximum than the second dye structure.

[0278] Item 126. The method according to any one of Items 118 to 125, wherein the first nucleic acid and the second nucleic acid consist of one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA).

[0279] Item 127. The method according to any one of items 118 to 126, wherein the base associated with each of the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid is selected from standard bases, modified bases and base analogs.

[0280] Item 128. The method according to Item 127, wherein at least one nucleotide of the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid is a nucleotide analog.

[0281] Item 129. The method according to any one of Items 118 to 128, wherein at least one nucleic acid probe further comprises a minor groove binding agent (MGB).

[0282] Item 130. The method according to Item 129, wherein the MGB is connected to the 3' end of the nucleic acid probe.

[0283] Item 131. The method according to any one of items 118 to 130, wherein the plurality of nucleotides in each of the first nucleic acid and the second nucleic acid comprises 10-50 nucleotides.

[0284] Item 132. The method according to Item 131, wherein each of the first nucleic acid and the second nucleic acid comprises 18-30 nucleotides.

[0285] Item 133. The method according to any one of items 118 to 132, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum ranging from 300 to 700 nm.

[0286] Item 134. The method according to any one of items 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 maximum values.

[0287] Item 135. The method according to Item 133 or 134, wherein the one or more excitable donor fluorophores have an excitation maximum ranging from 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.

[0288] Item 136. The method according to Item 135, wherein the one or more excitable donor fluorophores have emission maximum values ​​ranging from 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.

[0289] Item 137. The method according to any one of items 118 to 136, wherein the one or more excitable donor fluorophores are selected from bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0290] Item 138. The method according to any one of items 118 to 137, wherein the one or more excitable donor fluorophores are bonded to a water-soluble group.

[0291] Item 139. The method according to Item 138, wherein the water-soluble group includes a nonionic water-soluble group.

[0292] Item 140. The method according to Item 139, wherein the nonionic water-soluble group comprises polyalkylene glycol.

[0293] Item 141. The method according to Item 140, wherein the polyalkylene glycol comprises polyethylene glycol.

[0294] Item 142. The method according to any one of items 118 to 141, wherein the maximum excitation range of the one or more acceptor fluorophores is 450 to 750 nm.

[0295] Item 143. The method according to any one of items 118 to 142, wherein the one or more receptor fluorophores associated with the first nucleic acid probe and the one or more receptor fluorophores associated with the second nucleic acid probe have different emission maximum values.

[0296] Item 144. The method according to Item 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.

[0297] Item 145. The method according to any one of items 118 to 144, wherein the one or more acceptor fluorophores are selected from coumarin dyes, cyanine dyes, rhodamine dyes, xanthan dyes, polyacetylenes, dipyrrole methyl boron difluoride, naphthalimide, thiazide dyes, and acridine dyes.

[0298] Item 146. The method according to any one of items 118 to 145, wherein in each of the first nucleic acid probe and the second nucleic acid probe, the number of donor fluorophores exceeds the number of recipient fluorophores.

[0299] Item 147. The method according to Item 146, wherein the number of donor fluorophores ranges from 2 to 20.

[0300] Item 148. The method according to any one of items 118 to 147, wherein the first dye structure and the second dye structure each comprise a single acceptor fluorophore.

[0301] Item 149. The method according to any one of Items 18 to 148, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptide-like substances, and polynucleotides.

[0302] Item 150. The method according to Item 149, wherein each of the non-conjugated polymer backbones comprises a peptide.

[0303] Item 151. The method according to Item 150, wherein the peptide comprises 5 to 100 amino acid residues.

[0304] Item 152. The method according to any one of items 118 to 151, wherein the quencher absorbs light at the emission maximum of the one or more acceptor fluorophores.

[0305] Item 153. The method according to any one of items 118 to 152, wherein the quencher absorbs light in the range of 450 to 875 nm.

[0306] Item 154. The method according to Item 153, wherein the quencher absorbs light in the range of 440 to 485 nm, 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0307] Item 155. The method according to any one of Items 118 to 154, wherein the quencher is selected from BlackHole Quencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher.

[0308] Item 156. The method according to any one of items 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, QXL 490, QXL 570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl) or tetramethylrhodamine (TAMRA).

[0309] Item 157. The method according to any one of items 118 to 156, wherein the first dye structure and the second dye structure are attached to the 5' ends of the first nucleic acid and the second nucleic acid, and the quencher is attached to the 3' ends of the first nucleic acid and the second nucleic acid.

[0310] Item 158. The method according to any one of items 118 to 156, wherein the first dye structure and the second dye structure are attached to the 3' ends of the first nucleic acid and the second nucleic acid, and the quencher is attached to the 5' ends of the first nucleic acid and the second nucleic acid.

[0311] Item 159. The method according to Item 157 or 158, wherein one or both of the first nucleic acid probe and the second nucleic acid probe further comprises a second quencher linked to a nucleotide located at 7 to 15 nucleotides away from the 5' end of the nucleic acid.

[0312] Item 160. The method according to any one of items 157 to 159, wherein the second quencher is linked to a nucleotide located at 9 or 10 nucleotides away from the 5' end of the nucleic acid.

[0313] Item 161. The method according to Item 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, QXL 490, QXL570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphorus amide quencher, azo quencher, or anthraquinone quencher.

[0314] Item 162. The method according to any one of Items 118 to 161, wherein the reaction mixture further comprises one or more of the following: a buffer, water, deoxynucleoside triphosphate, nucleoside triphosphate, polymerase, 5'-3' nuclease, and polymerase having 5'-3' nuclease activity.

[0315] Item 163. A nucleic acid comprising: The first and second nucleic acids each contain multiple nucleotides that form a chain from the 5' end to the 3' end. Wherein, at least a portion of the first nucleic acid is complementary to the second nucleic acid, and The first nucleic acid comprises: A dye structure attached to the first end of the first nucleic acid, wherein the dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the first nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship.

[0316] Item 164. The nucleic acid according to Item 163, wherein the first nucleic acid comprises one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA).

[0317] Item 165. The nucleic acid according to Item 163 or 164, wherein the base associated with each of the plurality of nucleotides of the first nucleic acid is selected from standard bases, modified bases and base analogs.

[0318] Item 166. The nucleic acid according to Item 165, wherein at least one of the plurality of nucleotides is a nucleotide analog.

[0319] Item 167. The nucleic acid according to any one of items 163 to 166, wherein the first nucleic acid further comprises a small groove adhesive (MGB) connected to the first nucleic acid.

[0320] Item 168. The nucleic acid according to Item 167, wherein the MGB is linked to the 3' end of the first nucleic acid.

[0321] Item 169. The nucleic acid according to any one of items 163 to 168, wherein the plurality of nucleotides comprises 10 to 50 nucleotides.

[0322] Item 170. Nucleic acid as described in Item 169, wherein the plurality of nucleotides comprises 18-30 nucleotides.

[0323] Item 171. The nucleic acid according to any one of items 163 to 170, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum ranging from 300 to 700 nm.

[0324] Item 172. The nucleic acid according to Item 171, 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.

[0325] Item 173. The nucleic acid according to Item 171 or 172, wherein the one or more excitable donor fluorophores have emission maximum values ​​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.

[0326] Item 174. The nucleic acid according to any one of items 163 to 173, wherein the one or more excitable donor fluorophores are selected from bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0327] Item 175. The nucleic acid according to any one of items 163 to 174, wherein one or more excitable donor fluorophores are bonded to a water-soluble group.

[0328] Item 176. The nucleic acid according to Item 175, wherein the water-soluble group includes a nonionic water-soluble group.

[0329] Item 177. Nucleic acid according to Item 176, wherein the nonionic water-soluble group comprises polyalkylene glycol.

[0330] Item 178. Nucleic acids according to Item 177, wherein the polyalkylene glycol comprises polyethylene glycol.

[0331] Item 179. The nucleic acid according to any one of items 163 to 178, wherein the maximum excitation range of the one or more receptor fluorophores is 450 to 750 nm.

[0332] Item 180. The nucleic acid according to Item 179, wherein the one or more receptor 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.

[0333] Item 181. The nucleic acid according to any one of items 163 to 180, wherein the maximum emission range of the one or more receptor fluorophores is 500 to 875 nm.

[0334] Item 182. The nucleic acid according to Item 181, wherein the one or more receptor fluorophores have emission maximum values ​​in the range of 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0335] Item 183. The nucleic acid according to any one of items 163 to 182, wherein the one or more receptor fluorophores are selected from coumarin dyes, cyanine dyes, rhodamine dyes, xanthan dyes, polyacetylenes, dipyrrole methyl boron difluoride, naphthalimide, thiazide dyes, and acridine dyes.

[0336] Item 184. The nucleic acid according to any one of items 163 to 183, wherein the number of donor fluorophores exceeds the number of recipient fluorophores.

[0337] Item 185. Nucleic acid according to item 184, wherein the number of donor fluorophores ranges from 2 to 20.

[0338] Item 186. The nucleic acid according to any one of items 163 to 185, wherein the dye structure comprises a single receptor fluorophore.

[0339] Item 187. The nucleic acid according to any one of items 163 to 186, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptide-like substances, and polynucleotides.

[0340] Item 188. Nucleic acid as described in Item 187, wherein the non-conjugated polymer backbone comprises a peptide.

[0341] Item 189. The nucleic acid as described in Item 188, wherein the peptide comprises 5 to 100 amino acid residues.

[0342] Item 190. The nucleic acid according to any one of items 163 to 189, wherein the quencher absorbs light at the emission maximum of the one or more receptor fluorophores.

[0343] Item 191. The nucleic acid according to any one of items 163 to 190, wherein the quencher absorbs light in the range of 450 to 875 nm.

[0344] Item 192. The nucleic acid according to Item 191, wherein the quencher absorbs light in the range of 440 to 485 nm, 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0345] Item 193. The nucleic acid according to any one of items 191 to 192, wherein the quencher is selected from BlackHole Quencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher.

[0346] Item 194. The nucleic acid according to Item 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, QXL 490, QXL 570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl) or tetramethylrhodamine (TAMRA).

[0347] Item 195. The nucleic acid according to any one of items 163 to 194, wherein the dye structure is attached to the 5' end of the first nucleic acid and the quencher is attached to the 3' end of the first nucleic acid.

[0348] Item 196. The nucleic acid according to any one of items 163 to 194, wherein the dye structure is attached to the 3' end of the first nucleic acid and the quencher is attached to the 5' end of the first nucleic acid.

[0349] Item 197. The nucleic acid according to Item 195 or 196 further includes a second quencher linked to a nucleotide located 7 to 15 nucleotides away from the 5' end of the first nucleic acid.

[0350] Item 198. The nucleic acid according to any one of items 195 to 197, wherein the second quencher is linked to a nucleotide located at 9 or 10 nucleotides away from the 5' end of the first nucleic acid.

[0351] Item 199. The nucleic acid according to Item 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, QXL 490, QXL570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphorus amide quencher, azo quencher, or anthraquinone quencher.

[0352] Item 200. A system for detecting nucleic acids, the system comprising: A room for storing samples; An illumination source oriented to illuminate the sample; and A detector, which is oriented such that light from the sample illuminates the detector; The sample contains a nucleic acid probe, and the nucleic acid probe comprises: A nucleic acid complementary to a target nucleic acid, wherein the nucleic acid comprises multiple nucleotides forming a chain from the 5' end to the 3' end. A dye structure attached to the first end of the nucleic acid, wherein the dye structure comprises: Non-conjugated polymer backbone, One or more donor fluorophores connected to the non-conjugated polymer backbone, and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship, and A quencher attached to the second end of the nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; and The sample contains or is suspected of containing the target nucleic acid.

[0353] Item 201. The system according to Item 200, wherein the chamber contains a heating element to change the temperature of the sample.

[0354] Item 202. The system according to Item 201, wherein the heating element comprises a water bath or a hot block.

[0355] Item 203. The system according to Item 200 or 201, wherein the heating element employs a thermoelectric effect to change the temperature of the sample.

[0356] Item 204. The system according to any one of items 200 to 203 further includes a user interface to allow selection of temperature, irradiation wavelength, detection wavelength, and temperature cycling program.

[0357] Item 205. A system according to any one of items 200 to 204, wherein the nucleic acid comprises one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA).

[0358] Item 206. The system according to any one of items 200 to 205, wherein the base associated with each nucleotide of the plurality of nucleotides is selected from standard bases, modified bases, and base analogs.

[0359] Item 207. The system according to Item 206, wherein at least one of the plurality of nucleotides is a nucleotide analog.

[0360] Item 208. The system according to any one of items 200 to 207, wherein the nucleic acid probe further comprises a small groove adhesive (MGB) attached to the nucleic acid probe.

[0361] Item 209. The system according to Item 208, wherein the MGB is connected to the 3' end of the nucleic acid probe.

[0362] Item 210. The system according to any one of items 200 to 209, wherein the plurality of nucleotides comprises 10 to 50 nucleotides.

[0363] Item 211. The system according to Item 210, wherein the plurality of nucleotides comprises 18-30 nucleotides.

[0364] Item 212. The system according to any one of items 200 to 211, wherein the one or more excitable donor fluorophores are selected to have an excitation maximum value ranging from 300 to 700 nm.

[0365] Item 213. The system according to Item 212, 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.

[0366] Item 214. The system according to Item 212 or 213, wherein the one or more excitable donor fluorophores have emission maximum values ​​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.

[0367] Item 215. The system according to any one of items 200 to 214, wherein the one or more excitable donor fluorophores are selected from bodipy chromophores, aryl or heteroaryl chromophores, coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0368] Item 216. The system according to any one of items 200 to 215, wherein the one or more excitable donor fluorophores are bonded to a water-soluble group.

[0369] Item 217. The system according to Item 216, wherein the water-soluble group comprises a nonionic water-soluble group.

[0370] Item 218. The system according to Item 217, wherein the nonionic water-soluble group comprises polyalkylene glycol.

[0371] Item 219. The system according to Item 218, wherein the polyalkylene glycol comprises polyethylene glycol.

[0372] Item 220. The system according to any one of items 200 to 219, wherein the maximum excitation range of the one or more acceptor fluorophores is 450 to 750 nm.

[0373] Item 221. The system according to Item 220, wherein the one or more acceptor fluorophores have an excitation maximum ranging from 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.

[0374] Item 222. The system according to any one of items 200 to 221, wherein the maximum emission range of the one or more acceptor fluorophores is 500 to 875 nm.

[0375] Item 223. The system according to Item 222, wherein the one or more acceptor fluorophores have emission maximum values ​​in the range of 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0376] Item 224. The system according to any one of items 200 to 223, wherein the one or more acceptor fluorophores are selected from coumarin dyes, cyanine dyes, rhodamine dyes, xanthan dyes, polyacetylenes, dipyrrole methyl boron difluoride, naphthalimide, thiazide dyes, and acridine dyes.

[0377] Item 225. The system according to any one of items 200 to 224, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

[0378] Item 226. The system according to Item 225, wherein the number of donor fluorophores ranges from 2 to 20.

[0379] Item 227. The system according to any one of items 200 to 226, wherein the dye structure comprises a single acceptor fluorophore.

[0380] Item 228. The system according to any one of items 200 to 227, wherein the non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, lipids, peptide-like substances, and polynucleotides.

[0381] Item 229. The system according to Item 228, wherein the non-conjugated polymer backbone comprises a peptide.

[0382] Item 230. The system according to Item 229, wherein the peptide comprises 5 to 100 amino acid residues.

[0383] Item 231. The system according to any one of items 200 to 230, wherein the quencher absorbs light at the emission maximum of the one or more acceptor fluorophores.

[0384] Item 232. The system according to any one of items 200 to 231, wherein the quencher absorbs light in the range of 450 to 875 nm.

[0385] Item 233. The system according to Item 232, wherein the quencher absorbs light in the range of 440 to 485 nm, 485 to 510 nm, 510 to 565 nm, 565 to 590 nm, 590 to 625 nm, 625 to 740 nm or 740 to 875 nm.

[0386] Item 234. The system according to Item 232 or 233, wherein the quencher is selected from Black HoleQuencher (BHQ), QSY quencher, Iowa Black quencher, or QXL quencher.

[0387] Item 235. The system according to Item 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, QXL 490, QXL 570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl) or tetramethylrhodamine (TAMRA).

[0388] Item 236. The system according to any one of items 200 to 235, wherein the dye structure is attached to the 5' end of the nucleic acid and the quencher is attached to the 3' end of the nucleic acid.

[0389] Item 237. The system according to any one of items 200 to 235, wherein the dye structure is attached to the 3' end of the nucleic acid and the quencher is attached to the 5' end of the nucleic acid.

[0390] Item 238. The system according to Items 236 or 237 further includes a second quencher linked to a nucleotide located 7 to 15 nucleotides away from the 5' end of the nucleic acid.

[0391] Item 239. The system according to any one of items 236 to 238, wherein the second quencher is linked to a nucleotide located at 9 or 10 nucleotides away from the 5' end of the nucleic acid.

[0392] Item 240. The system according to Item 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, QXL 490, QXL570, QXL 670, 4-(dimethylamino)-azobenzene-4'-carboxylic acid (Dabcyl), tetramethylrhodamine (TAMRA), phosphorus amide quencher, azo quencher, or anthraquinone quencher.

[0393] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically not feasible. Those skilled in the art will understand that various other omissions, additions, and modifications may be made to the methods and structures described above 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 defined by the appended claims.

[0394] Those skilled in the art will understand that, in general, the terms used herein, and especially those used in the appended claims (e.g., the body of the appended claims), are intended to be “open” terms (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “including” should be interpreted as “including but not limited to,” etc.). Those skilled in the art will also understand that if the intent is to introduce a specific number of claim statements, then such intent will be explicitly stated in the claims, and without such a statement, such intent does not exist. For example, to aid understanding, the appended claims may contain the use of the introductory phrases “at least one” and “one or more” to introduce claim statements. However, the use of such phrases should not be construed as implying that introducing a claim statement with the indefinite article “a” or “an” limits any particular claim containing such an introduced claim statement to an embodiment containing only one such statement, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted as “at least one” or “one or more”); the same applies to the use of definite articles to introduce claim statements. Furthermore, even if the specific number of introduced claim statements is explicitly stated, those skilled in the art will recognize that such a statement should be interpreted as indicating at least the number of statements (e.g., the simple statement “two statements” without other modifiers means at least two statements, or two or more statements). Furthermore, in cases where conventions such as "at least one of A, B, and C" are used, this construction is generally intended to ensure that a person skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems with only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Additionally, in cases where conventions such as "at least one of A, B, or C" are used, this construction is generally intended to ensure that a person skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems with only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). A person skilled in the art should also understand that any transition words and / or phrases presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one, any, or both terms. For example, the phrase “A or B” would be understood to include the possibility of “A” or “B” or “A and B”.

[0395] Furthermore, when the features or aspects of this disclosure are described in accordance with the Markush Group, those skilled in the art will recognize that this disclosure is therefore also described in accordance with any individual member or subgroup of the Markush Group.

[0396] As those skilled in the art will understand, for any and all purposes, such as providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily considered sufficiently descriptive and such that the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. As those skilled in the art will also understand, all language such as “up to,” “at least,” “greater than,” “less than,” and similar terms includes the listed numbers and refers to a scope that can subsequently be decomposed into subscopes as described above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 items means a group having 1, 2, or 3 items. Similarly, a group having 1-5 items means a group having 1, 2, 3, 4, or 5 items, and so on.

[0397] Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0398] Therefore, the foregoing only illustrates the principles of the invention. It should be understood that those skilled in the art will be able to conceive of various arrangements, although not explicitly described or shown herein, which embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recorded herein are primarily intended to assist the reader in understanding the principles of the invention and the concepts contributed by the inventors to the field, and should be interpreted as not being limited to such specifically recorded examples and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of the invention and their specific examples are intended to cover their structural and functional equivalents. Additionally, such equivalents are intended to include both currently known equivalents and future development equivalents, i.e., any element developed that performs the same function regardless of its structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly recited in the claims.

[0399] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is explicitly defined as being invoked only when the exact phrase “means for” or the exact phrase “step for” is recited at the beginning of a definition in the claim; if such an exact phrase is not used in the definition in the claim, then 35 USC §112(f) or 35 USC §112(6) is not invoked.

Claims

1. A probe for nucleic acid detection, the probe comprising: Nucleic acids that are complementary to the target sequence, among which, The nucleic acid comprises multiple nucleotides that form a chain from the 5' end to the 3' end; A dye structure attached to the first end of the nucleic acid, wherein the dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the nucleic acid, wherein the quencher and one or more receptor fluorophores are in an energy transfer relationship.

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

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

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

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

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

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

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

9. The probe according to any one of the preceding claims, wherein, The non-conjugated polymer backbone comprises at least one of peptides, carbohydrates, hydrocarbons, polyethylene glycol (PEG) polymers, lipids, peptide-like substances, and polynucleotides.

10. The probe according to claim 9, wherein, The non-conjugated polymer backbone contains peptides.

11. A probe set for nucleic acid detection, the set comprising: A first nucleic acid probe, the first nucleic acid probe comprising: The first nucleic acid complementary to the first target sequence, wherein, The first nucleic acid comprises a plurality of nucleotides, which form a chain from the 5' end to the 3' end; A first dye structure attached to the first end of the first nucleic acid, wherein the first dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the first nucleic acid, wherein the quencher and the receptor fluorophore exhibit an energy transfer relationship; and A second nucleic acid probe, the second nucleic acid probe comprising: A second nucleic acid complementary to a second target sequence, wherein the second nucleic acid comprises a plurality of nucleotides forming a chain from the 5' end to the 3' end; A second dye structure attached to the first end of the second nucleic acid, wherein the second dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the second nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; Wherein, the first target sequence is different from the second target sequence; and The first dye structure has a different emission maximum value than the second dye structure.

12. A kit for nucleic acid detection, the kit comprising: A first nucleic acid probe, the first nucleic acid probe comprising: The first nucleic acid complementary to the first target sequence, wherein, The first nucleic acid comprises a plurality of nucleotides, which form a chain from the 5' end to the 3' end; A first dye structure attached to the first end of the first nucleic acid, wherein the first dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the first nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship.

13. A method for determining the presence of a target nucleic acid in a sample, the method comprising: (a) Generating a reaction mixture, the reaction mixture comprising: The sample; and Nucleic acid probe, the nucleic acid probe comprising: A nucleic acid complementary to the target nucleic acid, wherein the nucleic acid comprises a plurality of nucleotides forming a chain from the 5' end to the 3' end. A dye structure attached to the first end of the nucleic acid, wherein the dye structure comprises: Non-conjugated polymer backbone, One or more donor fluorophores connected to the non-conjugated polymer backbone, and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship, and A quencher attached to the second end of the nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; and Wherein, the nucleic acid probe hybridizes with any copy of the target nucleic acid in the sample; and (b) Monitoring the reaction mixture in response to a signal emitted from the dye structure to determine the presence of the target nucleic acid in the sample.

14. A nucleic acid, said nucleic acid comprising: The first and second nucleic acids each contain multiple nucleotides that form a chain from the 5' end to the 3' end. in, The first nucleic acid is complementary to at least a portion of the second nucleic acid, and The first nucleic acid comprises: A dye structure attached to the first end of the first nucleic acid, wherein the dye structure comprises: Non-conjugated polymer backbone; One or more donor fluorophores connected to the non-conjugated polymer backbone; and One or more acceptor fluorophores are attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore have an energy transfer relationship; and A quencher attached to the second end of the first nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship.

15. A system for detecting nucleic acids, the system comprising: A room for storing samples; An irradiation source oriented to irradiate the sample; as well as A detector, which is oriented such that light emitted from the sample illuminates the detector; in, The sample contains a nucleic acid probe, wherein the nucleic acid probe comprises: A nucleic acid complementary to a target nucleic acid, wherein the nucleic acid comprises a plurality of nucleotides forming a chain from the 5' end to the 3' end. A dye structure attached to the first end of the nucleic acid, wherein the dye structure comprises: A non-conjugated polymer backbone, one or more donor fluorophores connected to the non-conjugated polymer backbone, and one or more acceptor fluorophores connected to the non-conjugated polymer backbone, wherein the donor fluorophores and the acceptor fluorophores have an energy transfer relationship. A quencher attached to the second end of the nucleic acid, wherein the quencher and the receptor fluorophore have an energy transfer relationship; and The sample contains or is suspected of containing the target nucleic acid.

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