Compositions, kits, and methods for detecting nucleic acids using in-channel multiplexing

Abstract

Compositions, kits, and methods are disclosed that enable in-channel multiplexing by enabling the determination of distinct detectable signals, each related to a different assay target, within the same detection channel. The plurality of detectable signals can be separately resolved and analyzed independently to enable detection and / or quantification of each respective target. By enabling the assay of multiple targets within the same detection channel, the multiplicity of multiplex assays is increased without the need for additional dyes or the problems associated with increased spectral overlap.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 433,546, filed on March 21, 2023; U.S. Provisional Patent Application No. 63 / 408,665, filed on September 21, 2022; U.S. Provisional Patent Application No. 63 / 356,863, filed on June 29, 2022; and U.S. Provisional Patent Application No. 63 / 356,874, filed on June 29, 2022, each of which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to compositions, kits, and methods that enable multiplexing by enabling the determination of signals associated with different assay targets that have similar or the same spectral characteristics. Aspects of the present disclosure further relate to compositions, kits, and methods that enable multiplexing by enabling the determination of signals associated with different assay targets using the same detection channel (e.g., within the same fluorescence channel).

[0003] Introduction Nucleic acid detection assays are often performed by adding a sample suspected of containing one or more target nucleic acids to a reaction mixture. The reaction mixture may contain one or more detectable labels, each designed to associate with a different target nucleic acid and generate a signal corresponding to the amount of target nucleic acid in the reaction mixture. In a "singleplex" assay, the reaction mixture contains a single detectable label designed to associate with a single target. Conversely, in a "multiplex" assay, the reaction mixture typically contains a plurality of different detectable labels, each designed to be specific for a different target nucleic acid. Thus, in a multiplex assay, multiple different targets in a single reaction mixture can be detected. In some applications, the detectable label is a fluorescent dye integrated with a nucleic acid probe, primer, or some other nucleic acid molecule designed to specifically hybridize with a corresponding target nucleic acid designed to associate therewith.

[0004] In various multiplex nucleic acid detection assays, detectable labels are assigned to different target nucleic acids respectively. Then, the presence and / or amount of each target nucleic acid can be determined by measuring the signal emitted from the detectable label in a separate "detection channel" corresponding to a specific characteristic of the corresponding emitted signal. For example, in the context of a fluorescent emission dye as a detectable label, the separate detection channels can correspond to the emission wavelength spectra associated with each dye. However, there can be a significant amount of overlap in the emission spectra of different dyes. As the overlap in the emission spectra increases, it becomes more difficult to resolve the separate detected emission (e.g., fluorescence) signals, and thus it becomes more difficult to detect and / or quantify each target. Excessive overlap can, for example, require complex deconvolution algorithms to adequately resolve the separate fluorescence signals.

[0005] Multiplexed dyes can be selected for the purpose of minimizing spectral overlap, but due to the finiteness of the emission spectrum, the number of distinct dyes that can be combined in the same multiplex assay without relying on a reaction protocol that becomes at least one level more complex and deconvolution requirements at the backend is practically limited. As a result, currently, there are significant constraints on the number of different targets that can be detected and / or measured in a multiplex assay. Accordingly, there remains a need for compositions, kits, and methods that can increase the "plexy" of detection assays. Further, it may be desirable to separately use dyes that have some degree of overlap in their emission spectra, and / or dyes that use the same dye for different target nucleic acids.

[0006] When performing multiplexing to determine the relative amounts of different target nucleic acids in a sample, several challenges can arise. In particular, using detectable labels with overlapping emission spectra can make it difficult to individually determine the contribution of each label and, by extension, the contribution of each of the different target nucleic acids to which they bind.

[0007] There is a need to provide more robust techniques for performing multiplex nucleic acid detection assays, such as nucleic acid detection using various polymerase chain reaction (PCR) assays.

Brief Description of the Drawings

[0008] When the following descriptions of various embodiments are interpreted in conjunction with the accompanying drawings and the scope of the appended claims (both of which form part of this specification), various objects, features, characteristics, and advantages of the present invention within the scope of the present disclosure will become apparent and be more readily understood. In the drawings, like reference numerals may be used to designate corresponding or similar parts in the various figures, and the various elements shown are not necessarily drawn to scale:

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Mode for Carrying Out the Invention

[0009] Selected list of defined terms In the context of a nucleic acid probe and a target nucleic acid, the term "specifically interacts" (and similar terms) means that the probe is designed to interact with the target to a greater extent than with non-target nucleic acids also present in the reaction mixture. For example, specific interaction can include overall or partial hybridization of the probe with its corresponding target. Hybridization between the probe and the target need not be 100%. For example, a functionally effective interaction can be achieved using a probe having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or up to 100% homology to each target.

[0010] As used herein, a "detection channel" is a particular subset of the entire range of possible values of a detectable signal. For example, if the detectable signal is a fluorescence signal, a detection channel (i.e., a fluorescence channel or a dye channel) can represent a wavelength band of a particular size. A detection channel can have a bandwidth size of about 10 - 60 nm, depending on, for example, characteristics of the instrument such as sensitivity and / or the desired signal resolution accuracy. A detection channel can further include discontinuous wavelengths or wavelength ranges. A detection channel can be defined in addition to or instead of the above, according to the optical filter arrangement used to measure the detectable signal. Each different detection channel typically comprises a particular optical filter arrangement for blocking emission other than that of the channel. Thus, as a functional definition, each detectable signal within a given optical filter arrangement can be considered to be within the same detection channel. Nevertheless, in some cases, different fluorescent labels (e.g., different chemical structures) are detected in the same detection channel. As an example, the fluorescent dyes Cy5 and Alexa647 emit similar emission wavelengths and can be detected within the same channel.

[0011] As used herein, "substantially identical" signals are signals that cannot be clearly distinguished from each other under the detection conditions used. Optionally, the emission spectra of two substantially identical signals overlap to such an extent that the individual signals cannot be detected, for example, when the composite emission spectrum does not show the presence of two distinct peaks. Optionally, "substantially identical fluorescence" emission can be within approximately the same wavelength band. For example, a first fluorescence signal and a second fluorescence signal having substantially identical fluorescence can have emission peaks that differ by about 10 nm or less, or about 8 nm or less, or about 6 nm or less, or about 4 nm or less, or about 2 nm or less, or about 1 nm or less, or cannot be substantially distinguished from each other by the detection equipment used to measure the fluorescence emission. In addition to, or alternatively to, this, fluorescence signals can be considered to have "substantially identical fluorescence" in applications where they are measured using the same detection apparatus, such as the same optical filter arrangement. In one embodiment, substantially identical signals have substantially identical excitation / absorption spectra and thus cannot be excited separately. Optionally, both labels are excited during detection. Both labels can be excited and / or detected simultaneously.

[0012] As used herein with respect to a signal, "substantial" indicates exceeding the background significantly. For example, a detectable signal having "substantial signal" and / or "substantial fluorescence" is a signal that significantly exceeds the background (i.e., baseline) level, including a fluorescence signal that significantly exceeds the background level / baseline level of fluorescence. This can be determined by a threshold that separates background fluorescence from substantial fluorescence. The threshold can vary according to the specific test protocol and application requirements. In some embodiments (e.g., embodiments without a passive reference), the threshold is set to ΔRn within a range having an endpoint defined by, for example, about 1,000 to about 30,000, or about 2,000 to about 20,000, or about 3,000 to about 15,000, or about 4,000 to about 6,000, or any two of the above values. In some embodiments (e.g., embodiments with a passive reference), the threshold is set to ΔRn of, for example, about 0.01 to 0.5. In some embodiments, the threshold is several percent higher than the baseline level, such as about 5 percent to about 10 percent higher than the baseline level.

[0013] The "background" level or "baseline" level of a signal (i.e., the background level / baseline level of fluorescence) during the amplification process can be determined according to methods known to those skilled in the art. As a non-limiting example, the baseline level can be determined as the median value of the signal of the amplification cycle before exponential amplification occurs. For example, exponential amplification can be determined when the change in the signal from one amplification cycle to the next exceeds a specific percentage indicating an exponential change.

[0014] As a result, signal levels and / or fluorescence levels that are not "substantial" according to the above may be described herein as "negligible." Similarly, with respect to probe binding, a probe "substantially binds" to its target when it binds significantly above background (e.g., above binding to non-targets). Optionally, at least 1%, 5%, 10%, 20%, 50%, or 80% of the probe or target binds.

[0015] As used herein, a "cleavable" probe is a probe that is cleaved as a result of a specific interaction between the probe and its respective target, and a corresponding label is released, resulting in an increase in the corresponding detectable signal.

[0016] As used herein, a "non-cleavable" probe is a probe having a label that is designed to remain associated with the probe throughout the assay. In a non-cleavable probe, the corresponding detectable signal changes not as a result of the release of the label from the probe, but in accordance with a change in the structure of the probe. Extendable fluorogenic probes, such as the universal or hairpin-type extendable fluorogenic probes described in various embodiments, are examples of non-cleavable probes.

[0017] The terms "detectable signal" and "label signal" are used synonymously herein. For example, a "first label signal" is a signal emitted by a first label of a first probe type, and a "second label signal" is a signal emitted by a second label of a second probe type. A "total signal" is the entire signal measured within a particular detection channel at a given time point or measurement point. Multiple different "detectable signals" / "label signals" may contribute to the same "total signal". For example, it may include a total signal, a signal generated by a first label of a first probe type, and a signal generated by a second label of a second probe type. In some embodiments, the signal is a fluorescence signal, and terms such as "first fluorescence signal", "second fluorescence signal", and "total fluorescence signal" may be used as specific examples of the corresponding broader terms.

[0018] The term "spectral similarity" refers to the emission signals of detectable labels having the same spectral profile or substantially overlapping spectral profiles. Thus, different probe types carrying the same detectable label, or different probe types carrying different detectable labels with substantial spectral overlap in their emission signals can both be regarded as probes having spectral similarity. In an embodiment, detectable labels having spectral similarity may be detectable in the same optical detection channel, but similarly, other techniques can also be used to detect the emission signals of such detectable labels. References to substantially overlapping spectra should be understood to mean spectral similarity.

[0019] As used herein, the term "endpoint" when referring to a cycle means a designated cycle at which the PCR process is considered to be complete and / or a designated cycle at which a signal threshold that exceeds the background signal by a predetermined amount occurs. In various embodiments, the endpoint cycle according to the present disclosure can be within the range of 20 to 45 cycles, for example, 30 to 40 cycles. However, the number of cycles up to the endpoint cycle can vary. For example, the number of cycles at the endpoint cycle can correlate with the point at which the luminescence (e.g., fluorescence) signal indicating the amplification product reaches approximately a plateau. Also, the "endpoint signal" refers to the luminescence signal measured at the endpoint cycle. The endpoint signal can be measured at any designated or selected cycle.

[0020] The chemical structures and chemical formulas described herein are constructed in accordance with the standard rules of chemical valency known in the chemical art.

[0021] When substituents are defined by their conventional chemical formulas written from left to right, they also include chemically identical substituents that result from writing the structure from right to left. For example, -CH2O is equivalent to -OCH2-.

[0022] The term "alkyl", by itself or as part of another substituent, unless otherwise specified, means a straight-chain (i.e., unbranched) or branched carbon chain (or carbon), or a combination thereof, which may be fully saturated, monounsaturated or polyunsaturated, and may include monovalent radicals, divalent radicals and polyvalent radicals. Alkyl can contain a specified number of carbons (e.g., C1 - C 10(which means 1 to 10 carbons). In embodiments, the alkyl is fully saturated. In embodiments, the alkyl is monounsaturated. In embodiments, the alkyl is polyunsaturated. The alkyl is an acyclic chain. Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers, such as homologs and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. An unsaturated alkyl group is a group having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl, 3-propynyl, 3-butynyl, and further higher molecular homologs and isomers. An alkoxy is an alkyl bonded to the remainder of the molecule via an oxygen linker (-O-). The alkyl portion can be an alkenyl portion. The alkyl portion can be an alkynyl portion. An alkenyl contains one or more double bonds. An alkynyl contains one or more triple bonds.

[0023] Unless otherwise specified, the term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkyl, exemplified by -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group has 1 to 24 carbon atoms, and in this specification, such groups having 10 or fewer carbon atoms are preferred. "Lower alkyl" or "lower alkylene" generally refers to a shorter-chain alkyl or alkylene group having 8 or fewer carbon atoms. The term "alkenylene" by itself or as part of another substituent means a divalent radical derived from an alkene, unless otherwise specified. The term "alkynylene" by itself or as part of another substituent means a divalent radical derived from an alkyne, unless otherwise specified. In embodiments, alkylene is fully saturated. In embodiments, alkylene is monounsaturated. In embodiments, alkylene is polyunsaturated. Alkenylene contains one or more double bonds. Alkynylene contains one or more triple bonds.

[0024] The term "heteroalkyl", by itself or in combination with another term, unless otherwise specified, means a stable straight-chain or branched-chain, or combinations thereof, containing at least one carbon atom and at least one heteroatom (e.g., O, N, Si, and S), wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) (e.g., N, S, Si, or P) may be located at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Heteroalkyl is an acyclic chain. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -S-CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. For example, up to two or three heteroatoms may be consecutive, such as -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl moiety may contain one heteroatom (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain two optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain three optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain four optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain five optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain up to eight optionally different heteroatoms (e.g., O, N, S, Si, or P). The term "heteroalkenyl", by itself or in combination with another term, unless otherwise specified, means a heteroalkyl containing at least one double bond. Heteroalkenyl may optionally contain, in addition to one or more double bonds, one or more double bonds and / or one or more triple bonds.The term "heteroalkynyl", by itself or in combination with another term, means a heteroalkyl containing at least one triple bond, unless otherwise specified. Heteroalkynyl may optionally contain, in addition to one or more triple bonds, one or more triple bonds and / or one or more double bonds. In embodiments, the heteroalkyl is fully saturated. In embodiments, the heteroalkyl is monounsaturated. In embodiments, the heteroalkyl is polyunsaturated.

[0025] Similarly, the term "heteroalkylene", by itself or as part of another substituent, unless otherwise specified, is not limited, but means a divalent radical derived from heteroalkyl exemplified by CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. Also, for the heteroalkylene group, a heteroatom may occupy one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Further, for the alkylene linking group and the heteroalkylene linking group, the orientation of the linking group is not indicated by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, a heteroalkyl group, when used herein, includes a group bonded to the rest of the molecule via a heteroatom such as -C(O)R', -C(O)NR', -NR'R'', -OR', -SR' and / or -SO2R'. When "heteroalkyl" is described and then a specific heteroalkyl group such as NR'R'' is described, the term heteroalkyl and the term -NR'R'' are understood to be neither overlapping nor mutually exclusive. Rather, the specific heteroalkyl group is described for clarity. Therefore, the term "heteroalkyl" should not be construed herein as excluding specific heteroalkyl groups such as -NR'R''. The term "heteroalkenylene", by itself or as part of another substituent, unless otherwise specified, means a divalent radical derived from heteroalkene. The term "heteroalkynylene", by itself or as part of another substituent, unless otherwise specified, means a divalent radical derived from heteroalkyne. In an embodiment, the heteroalkylene is fully saturated. In an embodiment, the heteroalkylene is monounsaturated. In an embodiment, the heteroalkylene is polyunsaturated. The heteroalkenylene contains one or more double bonds. The heteroalkynylene contains one or more triple bonds.

[0026] The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, unless otherwise specified, each mean a cyclic form of "alkyl" and "heteroalkyl", respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Further, in the case of heterocycloalkyl, the heteroatom can occupy the position where the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. "Cycloalkylene" and "heterocycloalkylene" mean divalent radicals derived from cycloalkyl and heterocycloalkyl, respectively, alone or as part of another substituent. In embodiments, the cycloalkyl is fully saturated. In embodiments, the cycloalkyl is monounsaturated. In embodiments, the cycloalkyl is polyunsaturated. In embodiments, the heterocycloalkyl is fully saturated. In embodiments, the heterocycloalkyl is monounsaturated. In embodiments, the heterocycloalkyl is polyunsaturated.

[0027] In embodiments, the term "cycloalkyl" means a monocyclic, bicyclic or polycyclic cycloalkyl ring system. In embodiments, the monocyclic system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, such a group can be saturated or unsaturated, but is not aromatic. In embodiments, the cycloalkyl group is fully saturated. The bicyclic or polycyclic cycloalkyl ring system refers to a plurality of rings fused together, where at least one of the fused rings is a cycloalkyl ring, and the plurality of rings are attached to the parent molecular moiety via any carbon atom contained within that cycloalkyl ring.

[0028] In an embodiment, the cycloalkyl is cycloalkenyl. The term "cycloalkenyl" is used according to its plain ordinary meaning. In an embodiment, the cycloalkenyl is a monocyclic, bicyclic or polycyclic cycloalkenyl ring system. A bicyclic cycloalkenyl ring system or a polycyclic cycloalkenyl ring system refers to a system in which a plurality of rings are fused together, where at least one of the fused rings is a cycloalkenyl ring, and the plurality of rings are attached to the parent molecular moiety through any carbon atom contained within that cycloalkenyl ring.

[0029] In an embodiment, the term "heterocycloalkyl" means a monocyclic, bicyclic or polycyclic heterocycloalkyl ring system. In an embodiment, the heterocycloalkyl group is fully saturated. A bicyclic heterocycloalkyl ring system or a polycyclic heterocycloalkyl ring system refers to a system in which a plurality of rings are fused together, where at least one of the fused rings is a heterocycloalkyl ring, and the plurality of rings are attached to the parent molecular moiety through any atom contained within that heterocycloalkyl ring.

[0030] The term "halo" or "halogen" by itself or as part of another substituent, unless otherwise indicated, means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. Further, terms such as "haloalkyl" are intended to include both monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0031] The term "acyl" means -C(O)R, where R is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl, unless otherwise indicated.

[0032] Unless otherwise indicated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent, which may be monocyclic, or fused together (i.e., fused-ring aryl), or a polycyclic (preferably 1 to 3 rings) covalently bonded. Fused-ring aryl refers to a plurality of rings fused together, where at least one of the fused rings is an aryl ring, and the plurality of rings are bonded to the parent molecular moiety through any carbon atom contained within the aryl ring. The term "heteroaryl" refers to an aryl group (or aryl ring) containing at least one heteroatom such as N, O, or S, where nitrogen and sulfur atoms are optionally oxidized and nitrogen atoms (if any) are optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl groups (i.e., a plurality of rings fused together, where at least one of the fused rings is an aromatic ring, and the plurality of rings are bonded to the parent molecular moiety through any atom contained within the aromatic ring). 5,6-fused-ring heteroarylene refers to a structure where two rings are fused together, where one ring has 5 members, the other ring has 6 members, and at least one ring is a heteroaryl ring. Similarly, 6,6-fused-ring heteroarylene refers to a structure where two rings are fused together, where one ring has 6 members, the other ring has 6 members, and at least one ring is a heteroaryl ring. Also, 6,5-fused-ring heteroarylene refers to a structure where two rings are fused together, where one ring has 6 members, the other ring has 5 members, and at least one ring is a heteroaryl ring. The heteroaryl group can be bonded to the rest of the molecule through a carbon or a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazolyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. Substituents for each of the above aryl ring systems and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene" each mean a divalent radical derived from aryl and heteroaryl, respectively, alone or as part of another substituent. The heteroaryl group substituent may be -O-bonded to the ring heteroatom nitrogen.

[0033] A spiro ring refers to two or more rings where adjacent rings are connected via a single atom. The individual rings within a spiro ring may be the same or different. The individual rings within a spiro ring may or may not be substituted and may have substituents different from those of other individual rings within a series of spiro rings. Substituents conceivable for the individual rings within a spiro ring are the same substituents conceivable for the same ring when it is not part of the spiro ring (e.g., substituents for a cycloalkyl ring or a heterocycloalkyl ring). A spiro ring may be a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heterocycloalkylene, and the individual rings within a spiro ring group may be any of the immediately preceding list, including having all rings of one type (e.g., all rings are substituted heterocycloalkylene and each ring may be the same or different substituted heterocycloalkylene). When referring to a spiro ring system, a heterocyclic spiro ring means a spiro ring where at least one ring is a heterocycle and each ring may be a different ring. When referring to a spiro ring system, a substituted spiro ring means that at least one individual ring is substituted and each substituent may optionally be different.

[0034] Symbol

[0035]

Chemical formula

[0036] As used herein, the term "oxo" means oxygen double-bonded to a carbon atom.

[0037] The term "alkylarylene" as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, an alkylarylene group has the following formula:

[0038]

Chem.

[0039] The alkyl arylene moiety may be substituted on the alkylene moiety or arylene linker (e.g., at carbon 2, carbon 3, carbon 4, or carbon 6) with a halogen, oxo, -N3, -CF3, -CCl3, -CBr3, -CI3, -CN, -CHO, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2CH3, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, a substituted or unsubstituted C1-C5 alkyl, or a substituted or unsubstituted 2-5 membered heteroalkyl) (e.g., with a substituent). In an embodiment, the alkyl arylene is unsubstituted.

[0040] The above terms (e.g., "alkyl", "heteroalkyl", "cycloalkyl", "heterocycloalkyl", "aryl", and "heteroaryl") each include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for the various radicals are provided below.

[0041] The substituents of alkyl radicals and heteroalkyl radicals (often referred to as groups such as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) can be, but are not limited to, -OR’, =O, =NR’, =N-OR’, -NR’R’’, -SR’, halogen, -SiR’R’’R’’’, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R’’, -OC(O)NR’R’’, -NR’’C(O)R’, -NR’C(O)NR’’R’’’, -NR’’C(O)2R’, -NRC(NR’R’’R’’’)=NR’’’’, -NRC(NR’R’’)=NR’’’, -S(O)R’, -S(O)2R’, -S(O)2NR’R’’, -NRSO2R’, -NR’NR’’R’’’, -ONR’R’’, -NR’C(O)NR’’NR’’’R’’’’, -CN, -NO2, -NR’SO2R’’, -NR’C(O)R’’, -NR’C(O)OR’’, -NR’OR’’, and can be one or more of various groups selected from a number in the range of 0 to (2m’+1), where m’ is the total number of carbon atoms in such a group. R, R’, R’’, R’’’ and R’’’’ are each preferably independently a hydrogen group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group (e.g., an aryl group substituted with 1 to 3 halogens), a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkyl group, an alkoxy group or a thioalkoxy group, or an arylalkyl group. For example, when the compounds described herein contain two or more R groups, the R groups are each independently selected, and the same is true for the R’, R’’, R’’’ and R’’’’ groups, when two or more of the R’ group, R’’ group, R’’’ group and R’’’’ group are present. When R’ and R’’ are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 4-membered, 5-membered, 6-membered or 7-membered ring. For example, -NR’R’’ includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.From the above considerations regarding substituents, one of ordinary skill in the art will understand that the term "alkyl" is intended to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, C(O)CH2OCH3, etc.).

[0042] Similar to the substituents described for alkyl radicals, the substituents for aryl groups and heteroaryl groups are diverse. For example, -OR’, -NR’R’’, -SR’, halogen, -SiR’R’’R’’’, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R’’, -OC(O)NR’R’’, -NR’’C(O)R’, -NR’C(O)NR’’R’’’, -NR’’C(O)2R’, -NR-C(NR’R’’R’’’)=NR’’’’, -NR-C(NR’R’’)=NR’’’, -S(O)R’, -S(O)2R’, -S(O)2NR’R’’, -NRSO2R’, -NR’NR’’R’’’, -ONR’R’’, -NR’C(O)NR’’NR’’’R’’’’, -CN, -NO2, -R’, -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl are selected in the range from 0 to the total number of open valences on the aromatic ring system, where R’, R’’, R’’’ and R’’’’ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. For example, when the compounds described herein contain two or more R groups, the R groups are each independently selected, and the same applies to the R’, R’’, R’’’ and R’’’’ groups, i.e., when two or more of the R’ groups, R’’ groups, R’’’ groups and R’’’’ groups are present, they are selected in the same manner.

[0043] Substituents on a ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene or heteroarylene) can be shown as substituents on the ring rather than on a specific atom of the ring (commonly referred to as a floating substituent). In such cases, the substituent may be attached to any of the ring atoms (in accordance with the rules of chemical valence), and in the case of a fused ring or spiro ring, a substituent shown as attached to one member of the fused ring or spiro ring (a floating substituent on a monocyclic ring) may be a substituent on any of the fused ring or spirocyclic rings (a floating substituent on a polycyclic ring). When the substituent is attached to the ring but not to a specific atom (a floating substituent) and the subscript of the substituent is an integer greater than 1, the multiple substituents may be on the same atom, the same ring, different atoms, different fused rings, different spiro rings, and each substituent may optionally be different. When the point of attachment of the ring to the remainder of the molecule is not limited to a single atom (a floating substituent), the point of attachment may be any atom of the ring, and in the case of a fused ring or spiro ring, may be any atom of either the fused ring or spiro ring while following the rules of chemical valence. When the ring, fused ring or spiro ring contains one or more ring heteroatoms and the ring, fused ring or spiro ring is shown with one or more floating substituents (not limited to, including the point of attachment to the remainder of the molecule), the floating substituent may be attached to the heteroatom. In a structure or formula having a floating substituent, when the ring heteroatom is shown as attached to one or more hydrogens (e.g., a ring nitrogen having two bonds to ring atoms and a third bond to a hydrogen), it will be understood that when the heteroatom is attached to a floating substituent, the substituent replaces the hydrogen while following the rules of chemical valence. When, in a structure or formula having a floating substituent, the ring heteroatom is shown as attached to one or more hydrogens (e.g., a ring nitrogen having two bonds to ring atoms and a third bond to a hydrogen), it will be understood that when the heteroatom is attached to a floating substituent, the substituent replaces the hydrogen while following the rules of chemical valence.

[0044] Two or more substituents may optionally be joined to form an aryl, heteroaryl, cycloalkyl or heterocycloalkyl group. Such so-called ring-forming substituents are typically, but not necessarily, attached to the cyclic basic structure. In one embodiment, the ring-forming substituents are attached to adjacent constituent atoms of the basic structure. For example, two ring-forming substituents attached to adjacent constituent atoms of the cyclic basic structure form a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single constituent atom of the basic structure. For example, two ring-forming substituents attached to a single constituent atom of the cyclic basic structure form a spiro ring structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent constituent atoms of the basic structure.

[0045] Two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR’) q -U-, where T and U are independently -NR-, -O-, -CRR’- or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -A-(CH2) r -B-, where A and B are independently -CRR’-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR’- or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be of the formula -(CRR’) s -X’-(C’’R’’R’’’) d- may be replaced by substituents, where s and d are independently integers from 0 to 3, and X’ is -O-, -NR’-, -S-, -S(O)-, -S(O)2- or -S(O)2NR’-. The substituents R, R’, R’’ and R’’’ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[0046] As used herein, the terms “heteroatom” or “ring heteroatom” are intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), selenium (Se) and silicon (Si). In embodiments, the terms “heteroatom” or “ring heteroatom” are intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si).

[0047] As used herein, “substituent” means a group selected from the following moieties: (A) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl or 2-4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl or 5-6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5-10 membered heteroaryl, 5-9 membered heteroaryl or 5-6 membered heteroaryl), and (B) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl or 2-4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl or 5-6 membered heterocycloalkyl), aryl (e.g., C6-C 10 aryl, C 10Aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl or 5- to 6-membered heteroaryl), substituted with at least one substituent selected from the following substituents: (i) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl or 5- to 6-membered heteroaryl), and (ii) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), which is substituted with at least one substituent selected from the following substituents: (a) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 aryl, C 10Aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl or 5- to 6-membered heteroaryl), and (b) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl or 5- to 6-membered heteroaryl) which is substituted with at least one substituent selected from the following substituents: oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 Aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl or 5- to 6-membered heteroaryl).

[0048] "Size-limited substituent" or "size-limited substituent group" when used herein means a group selected from all of the substituents described above for "substituent group", where substituted or unsubstituted alkyl is each substituted or unsubstituted C1-C substituent) or "size-limited substituent group" group) means, when used herein, a group selected from all of the substituents described above for "substituent group", where substituted or unsubstituted alkyl is each substituted or unsubstituted C1-C 20 alkyl, substituted or unsubstituted heteroalkyl is each substituted or unsubstituted 2-membered to 20-membered heteroalkyl, substituted or unsubstituted cycloalkyl is each substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted heterocycloalkyl is each substituted or unsubstituted 3-membered to 8-membered heterocycloalkyl, substituted or unsubstituted aryl is each substituted or unsubstituted C6-C 10 aryl, and substituted or unsubstituted heteroaryl is each substituted or unsubstituted 5- to 10-membered heteroaryl.

[0049] "Lower substituent" or "lower substituent group", as used herein, means a group selected from all of the substituents described above for "substituent group", where substituted or unsubstituted alkyls are each substituted or unsubstituted C1-C8 alkyls, substituted or unsubstituted heteroalkyls are each substituted or unsubstituted 2- to 8-membered heteroalkyls, substituted or unsubstituted cycloalkyls are each substituted or unsubstituted C3-C7 cycloalkyls, substituted or unsubstituted heterocycloalkyls are each substituted or unsubstituted 3- to 7-membered heterocycloalkyls, substituted or unsubstituted aryls are each substituted or unsubstituted phenyls, and substituted or unsubstituted heteroaryls are each substituted or unsubstituted 5- to 6-membered heteroaryls.

[0050] In some embodiments, each of the substituents described in the compounds herein is substituted with at least one substituent. More specifically, in some embodiments, the substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein are each substituted with at least one substituent. In other embodiments, at least one or all of these groups are substituted with at least one size-restricted substituent. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent.

[0051] In other embodiments of the compounds herein, substituted or unsubstituted alkyls are each substituted or unsubstituted C1-C 20may be alkyl, and each of the substituted or unsubstituted heteroalkyls is a substituted or unsubstituted 2-membered to 20-membered heteroalkyl, each of the substituted or unsubstituted cycloalkyls is a substituted or unsubstituted C3-C8 cycloalkyl, each of the substituted or unsubstituted heterocycloalkyls is a substituted or unsubstituted 3-membered to 8-membered heterocycloalkyl, each of the substituted or unsubstituted aryls is a substituted or unsubstituted C6-C 10 aryl, and / or each of the substituted or unsubstituted heteroaryls is a substituted or unsubstituted 5- to 10-membered heteroaryl. In some embodiments of the compounds herein, each of the substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C 20 alkylene, each of the substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2-membered to 20-membered heteroalkylene, each of the substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each of the substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3-membered to 8-membered heterocycloalkylene, each of the substituted or unsubstituted arylene is a substituted or unsubstituted C6-C 10 arylene, and / or each of the substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 10-membered heteroarylene.

[0052] In some embodiments, each of the substituted or unsubstituted alkyls is a substituted or unsubstituted C1-C8 alkyl, each of the substituted or unsubstituted heteroalkyls is a substituted or unsubstituted 2-membered to 8-membered heteroalkyl, each of the substituted or unsubstituted cycloalkyls is a substituted or unsubstituted C3-C7 cycloalkyl, each of the substituted or unsubstituted heterocycloalkyls is a substituted or unsubstituted 3-membered to 7-membered heterocycloalkyl, each of the substituted or unsubstituted aryls is a substituted or unsubstituted C6-C 10Aryl and / or substituted or unsubstituted heteroaryl are each a substituted or unsubstituted 5- to 9-membered heteroaryl. In some embodiments, substituted or unsubstituted alkylene are each a substituted or unsubstituted C1-C8 alkylene, substituted or unsubstituted heteroalkylene are each a substituted or unsubstituted 2- to 8-membered heteroalkylene, substituted or unsubstituted cycloalkylene are each a substituted or unsubstituted C3-C7 cycloalkylene, substituted or unsubstituted heterocycloalkylene are each a substituted or unsubstituted 3- to 7-membered heterocycloalkylene, and substituted or unsubstituted arylene are each a substituted or unsubstituted C6-C 10 Aryl and / or substituted or unsubstituted heteroaryl are each a substituted or unsubstituted 5- to 9-membered heteroaryl. In some embodiments, the compound is a chemical species described in the Examples section, drawings or tables below.

[0053] In an embodiment, the substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene and / or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., each is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene and / or unsubstituted heteroarylene). In an embodiment, the substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene and / or substituted or unsubstituted heteroarylene) is substituted (e.g., each is substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene).

[0054] In embodiments, the substituent moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, and when the substituent moiety is substituted with a plurality of substituents, the substituents may each optionally be different. In embodiments, when the substituent moiety is substituted with a plurality of substituents, the substituents are each different.

[0055] In embodiments, the substituent moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent having a limited size, and when the substituent moiety is substituted with a plurality of substituents having a limited size, the substituents having a limited size may each optionally be different. In embodiments, when the substituent moiety is substituted with a plurality of substituents having a limited size, the substituents having a limited size are each different.

[0056] In embodiments, the substituent moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one lower substituent, and when the substituent moiety is substituted with a plurality of lower substituents, the lower substituents may each optionally be different. In embodiments, when the substituent moiety is substituted with a plurality of lower substituents, the lower substituents are each different.

[0057] In embodiments, the substituent moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, a substituent with a restricted size or a lower substituent, and when the substituent moiety is substituted with a plurality of groups selected from substituents, substituents with a restricted size and lower substituents, the substituents, substituents with a restricted size and / or lower substituents may each optionally be different. In embodiments, when the substituent moiety is substituted with a plurality of groups selected from substituents, substituents with a restricted size and lower substituents, the substituents, substituents with a restricted size, and / or lower substituents are each different.

[0058] Certain compounds of the present disclosure have an asymmetric carbon atom (an optical or chiral center) or a double bond and can be defined as (R)- or (S)-, or (D)- or (L)- with respect to an amino acid from the perspective of enantiomers, racemates, diastereomers, tautomers, geometric isomers, and absolute stereochemistry. Stereoisometric forms, and individual isomers are included within the scope of the present disclosure. The compounds of the present disclosure do not include those known in the art to be too unstable to synthesize and / or isolate. The present disclosure is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-isomers, or (D)- and (L)-isomers can be prepared using a chiral synthon or chiral reagent or resolved using conventional techniques. When the compounds described herein contain an olefinic bond or other geometrically asymmetric center, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0059] As used herein, the term "isomer" refers to a compound that has the same number and same types of atoms and thus the same molecular weight, but differs in the structural or spatial arrangement of the atoms.

[0060] As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily convertible from one isomer to another.

[0061] It will be apparent to those skilled in the art that the specific compounds of the present disclosure may exist as tautomers, and that all such tautomers of the compounds are within the scope of the present disclosure.

[0062] Unless otherwise indicated, the structures shown herein are also intended to include all stereochemical forms of that structure, i.e., the R and S configurations for each asymmetric center. Thus, single stereoisomers of the compounds, as well as mixtures of enantiomers and mixtures of diastereomers, are within the scope of the present disclosure.

[0063] Unless otherwise indicated, the structures shown herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, substitution of hydrogen with deuterium or tritium, or substitution of carbon with 13 C or 14 carbon enriched in

[0064] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes in one or more of the atoms that make up such compounds. For example, the compounds may be radiolabeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All variations of isotopes of the compounds of the present disclosure are included within the scope of the present disclosure, whether radioactive or not.

[0065] Throughout this application, it should be noted that alternatives are written at each amino acid position that includes a Markush group, e.g., two or more possible amino acids. Each component of the Markush group is considered separately, whereby another embodiment is contemplated and the Markush group should not be construed as a single unit.

[0066] As used herein, the terms “bioconjugate” and “bioconjugate linker” refer to an association that occurs between atoms or molecules of a bioconjugate reactive group or a bioconjugate reactive moiety. The association may be direct or indirect. For example, a conjugate between a first bioconjugate reactive group (e.g., -NH2, -COOH, -N-hydroxysuccinimide or -maleimide) provided herein and a second bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amino acid containing an amine side chain or carboxylate) may be direct, for example, by a covalent bond or a linker (e.g., a first linker of a second linker), or may be indirect, for example, by a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (π effect), hydrophobic interactions, etc.). In embodiments, the bioconjugate or bioconjugate linker is formed using bioconjugate chemistry (i.e., the association of two bioconjugate reactive groups), including, but not limited to, nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reaction), and addition to carbon-carbon multiple bonds and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). In embodiments, a first bioconjugate reactive group (e.g., a maleimide moiety) is covalently bonded to a second bioconjugate reactive group (e.g., sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., a haloacetyl moiety) is covalently bonded to a second bioconjugate reactive group (e.g., sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., a pyridyl moiety) is covalently bonded to a second bioconjugate reactive group (e.g., sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., an -N-hydroxysuccinimide moiety) is covalently bonded to a second bioconjugate reactive group (e.g., an amine).In an embodiment, the first bioconjugate reactive group (e.g., a maleimide moiety) is covalently bonded to the second bioconjugate reactive group (e.g., a sulfhydryl). In an embodiment, the first bioconjugate reactive group (e.g., a - sulfo - N - hydroxysuccinimide moiety) is covalently bonded to the second bioconjugate reactive group (e.g., an amine).

[0067] Useful bioconjugate reactive moieties for the bioconjugate chemistry of this specification include, for example, the following: (a) without limitation, carboxyl groups and various derivatives thereof, including N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters; (b) hydroxyl groups that can be converted to esters, ethers, aldehydes, etc.; (c) haloalkyl groups in which the halide can be substituted, for example, later with a nucleophilic group such as an amine, carboxylate anion, thiolate anion, carbanion, or alkoxide ion, whereby a covalent bond of a new group can occur at the site of the halogen atom; (d) dienophile groups that can participate in the Diels-Alder reaction, for example, maleimide or maleimide groups; (e) aldehyde groups or ketone groups in which later derivatization is possible, for example, via the formation of carbonyl derivatives such as imines, hydrazones, semicarbazones, or oximes, or via mechanisms such as Grignard addition or alkyllithium addition; (f) sulfonyl halide groups that can later be reacted with an amine to form, for example, a sulfonamide; (g) thiol groups that can be converted to disulfides, react with acyl halides, bind to metals such as gold, or react with maleimide; (h) amine groups or sulfhydryl groups (for example, those present in cysteine) that can be acylated, alkylated, or oxidized, for example; (i) alkenes that can undergo, for example, addition cyclization, acylation, Michael addition, etc.; (j) epoxides that can react with, for example, amines and hydroxyl compounds; (k) phosphoramidites and other standard functional groups useful for nucleic acid synthesis; (l) metal-oxidized silicon bonds; (m) metals that bind to reactive phosphorus groups (for example, phosphines) to form, for example, phosphodiester bonds; (n) azides coupled to alkynes using copper-catalyzed addition cyclization click chemistry; and (o) biotin conjugates can react with avidin or streptavidin to form an avidin-biotin complex or a streptavidin-biotin complex.

[0068] The bioconjugate reactive group can be selected so as not to participate in or interfere with the chemical stability of the conjugate described herein. Alternatively, the presence of a protecting group can prevent the reactive functional group from participating in the crosslinking reaction. In an embodiment, the bioconjugate includes a molecular entity resulting from the reaction of an unsaturated bond such as maleimide with a sulfhydryl group.

[0069] "Analog", "analogue" or "derivative" is used according to its plain ordinary meaning in chemistry and biology, and is structurally similar to another compound (i.e., the so-called "reference" compound), but has a different composition, for example, in that one atom is replaced by an atom of a different element, or in that a particular functional group is present, or in that one functional group is replaced by another functional group, or in that the absolute stereochemistry of one or more chiral centers of the reference compound is different. Thus, an analog is a compound that is similar or equivalent to the reference compound in function and appearance, but not in structure or origin.

[0070] As used herein, the term "a" or "an" means one or more. Further, as used herein, the phrase "substituted with [n]" means that a particular group may be substituted with one or more of any or all of the specified substituents. For example, when a group such as an alkyl group or a heteroaryl group is "substituted with unsubstituted C1-C 20 alkyl or unsubstituted 2- to 20-membered heteroalkyl", the group may include one or more unsubstituted C1-C 20 alkyl and / or one or more unsubstituted 2- to 20-membered heteroalkyl.

[0071] Further, when a moiety is substituted with an R substituent, the group can be referred to as “R-substituted”. When a moiety is R-substituted, the moiety is substituted with at least one R substituent, and each R substituent may optionally be different. When a specific R group is present in the description of a chemical genus (e.g., formula (I)), Roman alphabet symbols may be used to distinguish the occurrences of the specific R group, respectively. For example, when multiple R 13 substituents are present, each R 13 substituent may be distinguished as R 13.A , R 13.B , R 13.C , R 13.D , etc., where R 13.A , R 13.B , R 13.C , R 13.D , etc. are each defined optionally differently within the scope of the definition of R 13 . When an R moiety, R group or R substituent disclosed herein is attached via a single bond representation and the R moiety, R group or R substituent is oxo, one of ordinary skill in the art will immediately recognize that the oxo is attached via a double bond in accordance with the normal rules of chemical valence.

[0072] The description of the compounds of the present disclosure is limited by the principles of chemical bonding known to those of ordinary skill in the art. Thus, when a group can be substituted by one or more of a number of substituents, such substitution is selected so as to be in accordance with the principles of chemical bonding, and not inherently unstable, and / or not likely to be unstable under ambient conditions such as aqueous, neutral and some known physiological conditions, as would be given by compounds known to those of ordinary skill in the art. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in accordance with the principles of chemical bonding known to those of ordinary skill in the art, thereby avoiding compounds that are inherently unstable.

[0073] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides), polymers thereof, or their complements, or nucleosides (e.g., deoxyribonucleosides or ribonucleosides) in any form of single-stranded, double-stranded, or multiple-stranded. In embodiments, "nucleic acid" does not include nucleosides. Terms such as "polynucleotide", "oligonucleotide", "oligo", etc. refer to nucleotides in a linear sequence in their usual and customary meanings. Oligonucleotides typically have a length of about 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 12 nucleotides, 15 nucleotides, 25 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides or more, up to about 100 nucleotides. Nucleic acids and polynucleotides are polymers of any length, including longer lengths such as 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. In certain embodiments, the nucleic acids herein contain phosphodiester bonds. In other embodiments, nucleic acid analogs with alternative backbones may be included, for example, those containing phosphoramidate bonds, phosphorothioate bonds, phosphorodithioate bonds, or O-methylphosphoramidite bonds, as well as nucleic acid analogs that may have the backbone and bonds of peptide nucleic acids. Other analog nucleic acids include those with a positively charged backbone, those with a non-ionic backbone, and those with a non-ribose backbone. Nucleic acids containing one or more carbocyclic sugars are also included in one definition of nucleic acids. Modifications of the ribose-phosphate backbone can be made for various reasons, for example, to increase the stability and half-life of such molecules in a physiological environment, or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be made, or mixtures of different nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs may also be made. The residues of nucleic acids referred to herein are monomers of nucleic acids (e.g., nucleotides). The term "nucleoside" refers to a glycosylamine containing a nucleobase and a pentose sugar (ribose or deoxyribose) in its usual and customary meanings.Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. Nucleosides may be modified in the base and / or sugar. The term "nucleotide" refers, in its ordinary and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single-stranded DNA and double-stranded DNA, single-stranded RNA and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and single-stranded and double-stranded RNA. Nucleic acids, e.g., examples of polynucleotides contemplated herein, include any type of RNA, e.g., mRNA, siRNA, miRNA, and guide RNA, and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "double-stranded" in the context of a polynucleotide refers, in its ordinary and customary sense, to double-strandedness. Nucleic acids can be linear or branched. For example, a nucleic acid can be a linear strand of nucleotides, or alternatively, a nucleic acid can be branched, e.g., to include one or more arms or branches of nucleotides. Optionally, a branched nucleic acid can be repeatedly branched to form higher-order structures such as dendrimers. As used herein, a "nucleic acid moiety" refers to a nucleic acid in its monovalent form. In embodiments, the nucleic acid moiety is attached to the 3' or 5' position of a nucleotide or nucleoside.

[0074] A nucleic acid (e.g., including a nucleic acid having a phosphorothioate backbone) can include one or more reactive moieties. As used herein, the term "reactive moiety" includes any group that can react with another molecule, e.g., a nucleic acid or polypeptide, via a covalent bond, non-covalent bond, or other interaction. By way of example, a nucleic acid can include an amino acid-reactive moiety that reacts with an amino acid on a protein or polypeptide via a covalent bond, non-covalent bond, or other interaction.

[0075] As used herein, "nucleotide" refers to a nucleoside-5'-phosphate (e.g., polyphosphate) compound or a structural analog thereof, which can be incorporated by a nucleic acid polymerase (e.g., partially incorporated as a nucleoside-5'-monophosphate or a derivative thereof) to extend a growing nucleic acid strand (such as a primer). A nucleotide may contain a base such as adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or an analog thereof, and may contain 1, 2, 3, 4, 5, 6, 7, 8 or more phosphates in the phosphate group. A nucleotide may be modified in one or more of the base, sugar or phosphate group. A nucleotide may be one to which a label or tag is attached ("labeled nucleotide" or "tagged nucleotide"). In embodiments, the nucleotide is a deoxyribonucleotide. In embodiments, the nucleotide is a ribonucleotide. In embodiments, the nucleotide contains three phosphate groups (e.g., triphosphate group).

[0076] This term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and non-natural, have binding properties similar to the reference nucleic acid, and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, for example, phosphoramidates, phosphorodiamidates, phosphorothioates (also known as phosphorothioates having a double-bond sulfur substituting for oxygen in the phosphate), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoglycolic acid, methyl phosphonate, boranophosphonate, or phosphodiester derivatives including O-methyl phosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), as well as modifications to nucleotide bases such as 5-methylcytidine or pseudouridine, and peptide nucleic acid backbones and linkages are non-limitingly mentioned. Other analog nucleic acids include those having a positively charged backbone, a non-ionic backbone, a modified sugar, and a non-ribose backbone (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNA: locked nucleic acid) known in the art). Nucleic acids containing one or more carbocyclic sugars are also included in one definition of nucleic acids. Modifications to the ribose-phosphate backbone can be made for various reasons, for example, to increase the stability and half-life of such molecules in the physiological environment or for use as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be made, or mixtures of different nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs can also be made. In an embodiment, the internucleotide linkages in DNA are phosphodiesters, phosphodiester derivatives, or a combination of both.

[0077] "Nucleoside" is structurally similar to nucleotide but lacks the phosphate moiety present in nucleotide. Examples of nucleoside analogs can be considered those in which a label is attached to the base and there is no phosphate group attached to the sugar molecule. As used herein, "nucleoside" refers to a glycosyl compound consisting of a nucleobase and a five-membered sugar (e.g., either ribose or deoxyribose). A nucleoside may contain a base such as adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or an analog thereof. The nucleoside may be modified in the base and / or and the sugar. In an embodiment, the nucleoside is a deoxyribonucleoside. In an embodiment, the nucleoside is a ribonucleoside.

[0078] As used herein, the terms "complementary" or "substantially complementary" refer to hybridization, base pairing, or duplex formation between nucleotides or nucleic acids. For example, complementarity is seen between the two strands of a double-stranded DNA molecule, or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid, when the nucleotides (e.g., RNA or DNA) or nucleotide sequences can base pair with their cognate nucleotides or cognate nucleotide sequences, respectively. As described herein and as generally known in the art, the complementary (matching) nucleotide of adenosine (A) is thymidine (T), and the complementary (matching) nucleotide of guanosine (G) is cytosine (C). Thus, a complement may include a sequence of nucleotides that base pairs with the corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of the complement may match the nucleotides of the second nucleic acid sequence partially or completely. When the nucleotides of the complement match each nucleotide of the second nucleic acid sequence completely, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. When the nucleotides of the complement match the nucleotides of the second nucleic acid sequence partially, only some of the nucleotides of the complement form base pairs with the nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and non-coding sequences, where the non-coding sequence contains the complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. Further examples of complementary sequences are sense and antisense sequences, where the sense sequence contains the complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence. A "duplex" means at least two oligonucleotides and / or polynucleotides that are completely or partially complementary, with Watson-Crick type base pairing occurring between all or most of their nucleotides, resulting in the formation of a stable complex.

[0079] As described herein, the complementarity of sequences may be partial, where only a portion of the nucleic acids match according to base pairing, or may be complete, where all of the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other may have complementary nucleotides at a particular percentage (e.g., about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more complementarity over a particular region). In embodiments, two sequences are complementary when they are completely complementary and have 100% complementarity.

[0080] As used herein, the term "polymerase" refers to any natural or non-natural enzyme or other catalyst that can catalyze polymerization reactions such as the polymerization of nucleotide monomers to form nucleic acid polymers. Exemplary types of polymerases that can be used in the compositions and methods of the present disclosure include DNA polymerases, DNA-dependent or RNA-dependent RNA polymerases, and reverse transcriptases. In some cases, the DNA polymerase is 9°N polymerase or a variant thereof, E. coli DNA polymerase I, bacteriophage T4 DNA polymerase, Sequenase, Taq DNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0 DNA polymerase, 9°N polymerase, 9°N polymerase (exo-) A485L / Y409V, Phi29 DNA polymerase (φ29 DNA polymerase), T7 DNA polymerase, DNA polymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNA polymerase V, VentR DNA polymerase, Therminator™ II DNA polymerase, Therminator™ III DNA polymerase, or Therminator™ IX DNA polymerase. As used herein, the terms "DNA polymerase" and "nucleic acid polymerase" are used according to their plain and ordinary meaning and refer to enzymes that can synthesize nucleic acid molecules from nucleotides (e.g., deoxyribonucleotides). Typically, DNA polymerases add nucleotides to the 3' end of a DNA strand, one nucleotide at a time.In an embodiment, the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Polβ DNA polymerase, Polμ DNA polymerase, Polλ DNA polymerase, Polσ DNA polymerase, Polα DNA polymerase, Pol δ DNA polymerase, Polε DNA polymerase, Polη DNA polymerase, Polι DNA polymerase, Polκ DNA polymerase, Polζ DNA polymerase, Polγ DNA polymerase, Polθ DNA polymerase, Polυ DNA polymerase, or a thermophilic nucleic acid polymerase (e.g., Therminatorγ, 9°N polymerase (exo-), Therminator II, Therminator III, or Therminator IX). In an embodiment, the DNA polymerase is a modified archaeal DNA polymerase. In an embodiment, the polymerase is a reverse transcriptase. In an embodiment, the polymerase is a mutant Pyrococcus abyssi (P.abyssi) polymerase (e.g., the mutant Pyrococcus abyssi (P.abyssi) polymerase described in International Publication No. 2018 / 148723 or International Publication No. 2020 / 056044). As used herein, the term "thermophilic nucleic acid polymerase" refers to a family of DNA polymerases (e.g., 9°N (trademark)) and variants thereof derived from DNA polymerases first isolated from hyperthermophilic archaea found in hydrothermal vents at that latitude of Thermococcus sp. 9°N - 7 (East Pacific Rise) (Southworth MW, et al. PNAS. 1996;93(11):5281 - 5285). Thermophilic nucleic acid polymerases are members of the family B DNA polymerases.

[0081] As used herein, the term "exonuclease activity" is used according to its ordinary meaning in the art and refers to the removal of nucleotides from a nucleic acid by a DNA polymerase. For example, during polymerization, nucleotides are added to the 3' end of the primer strand. A DNA polymerase may incorporate an incorrect nucleotide at the 3'-OH end of the primer strand, in which case the incorrect nucleotide cannot form a hydrogen bond with the corresponding base in the template strand. Such incorrectly added nucleotides are removed from the primer as a result of the 3'→5' exonuclease activity of the DNA polymerase. When referring to 3'-5' exonuclease activity, it is understood that the DNA polymerase promotes a hydrolysis reaction that cleaves the phosphodiester bond at the 3' end of the polynucleotide strand to excise the nucleotide. In embodiments, 3'-5' exonuclease activity refers to continuously removing nucleotides in a single-stranded DNA in the 3'→5' direction and sequentially releasing deoxyribonucleoside 5'-monophosphates.

[0082] The terms "determine," "calculate," and "estimate" are used interchangeably herein. These terms are not intended to imply a precise level of measurement accuracy. Thus, when a value is "determined," "calculated," or "estimated" using the embodiments described herein, it is understood that such a value may include inherent errors due to factors such as the tolerance of the detection device, rounding, variations in chemical reactions, and other inherent measurement imperfections known and understood by those of skill in the art.

[0083] Furthermore, for any given element of a component of the described embodiments, unless otherwise stated implicitly or explicitly, any of the possible alternatives listed for that element or component may generally be used individually, or in combination with each other.

[0084] In addition, unless otherwise indicated, numbers expressing quantities, ingredients, distances, or other measurements used in the specification and claims are to be understood as optionally modified by the term "about" or its synonyms. When terms such as "about," "approximately," "substantially," etc. are used with a recited quantity, value, or condition, it can be interpreted to mean a quantity, value, or condition that deviates by less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% from the recited quantity, value, or condition. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the reported number of significant digits and by applying ordinary rounding techniques.

[0085] The headings and subheadings used herein are for structural purposes only and are not intended to be used to limit the description or the scope of the claims.

[0086] It should also be noted that, as used in the specification and the scope of the appended claims, the singular forms "a," "an," and "the" do not exclude a plurality of referents unless the context clearly dictates otherwise. Thus, for example, embodiments referring to a singular referent (e.g., "widget") can also include two or more such referents.

[0087] The embodiments described herein may include the properties and / or characteristics (e.g., components, constituents, members, elements, parts, and / or portions) described in one or more separate embodiments, and it will be understood that they are not necessarily strictly limited to the features explicitly described for that particular embodiment. Thus, the various features of a given embodiment can be combined with, and / or incorporated into, other embodiments of the present disclosure. Accordingly, the disclosure of a particular feature associated with a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of that feature to that particular embodiment. Rather, it will be understood that other embodiments can also include such features.

[0088] Overview of multiplexing using probes having labels with spectral similarity FIG. 1A shows the emission spectra of various fluorescent dyes that can be used in nucleic acid detection assays. As noted above, in some multiplex assays, each dye is assigned to a separate target, and then the presence and / or amount of each target is determined, for example, by measuring the fluorescence signal in a separate detection channel corresponding to the emission wavelength of the corresponding dye. As shown in FIG. 1B, in some cases, a significant amount of overlap is seen in the emission spectra of the dyes. For example, dye 1 and dye 2 in FIG. 1B exhibit substantially overlapping emission spectra, which are considered to be detectable by the same channel (channel 5 in FIG. 1B). Multiplexing dyes are typically selected for the purpose of minimizing spectral overlap, but due to the finiteness of the emission spectra, the number of separate dyes that can be combined in the same multiplex assay when using existing techniques is practically limited, and thus the number of different targets that can be detected and / or measured is limited.

[0089] Embodiments described herein address one or more of the above problems by providing multiple detectable signals that are each associated with a different assay target or a series of targets and have similar emission spectra, e.g., that can correspond to detection in the same detection channel. The multiple detectable signals can be resolved separately and analyzed independently, thereby enabling detection and / or quantification of each target. By enabling assays of multiple targets using the same dye or dyes having spectral similarity, the disclosed embodiments can beneficially increase the “plexy” of multiplex assays (i.e., the number of targets that can be detected and quantified) without reliance on additional dyes or problems associated with increased spectral overlap. For example, in accordance with aspects of the present disclosure, dyes having spectral similarity but directed to different target analytes can be detected using a common detection channel. Similarly, embodiments described herein can beneficially reduce the number of distinct dyes required for a multiplex assay without reducing the plexy of the assay. Further, various embodiments can enable use of the same dye as a label for different target nucleic acids in a multiplex assay, including simultaneous use of the same dye for different targets during a reaction. Still further, various embodiments can enable detection of the same dye in the same detection channel.

[0090] FIG. 2A is a schematic diagram of a method for detecting multiple target nucleic acids using detectable labels having spectral similarity by preparing different first and second probe types, varying the conditions of the reaction mixture, and measuring the total signal obtained under each set of conditions. As shown, the first probe 202 is designed to specifically interact ( "bind") with the first target 206. The first probe 202 includes a first label 210 that can generate a first labeled signal 214. The second probe 204 is designed to specifically interact with a second target 208 that is different from the first target 206. The second probe 204 includes a second label 212 that can generate a second labeled signal 216.

[0091] In some embodiments, the first label 210 and the second label 212 are the same. For example, the first label 210 and the second label 212 may include the same fluorescent dye. In some embodiments, the first label 210 and the second label 212 may be different, but are still designed to generate substantially the same signal (e.g., having spectral similarity). For example, the first label 210 and the second label 212 may include dyes that are chemically different but function to emit fluorescent signals having similar wavelengths. In some embodiments, the first labeled signal 214 and the second labeled signal 216 are measured using the same detection channel (e.g., one including an optical filter arrangement) within the detection device.

[0092] The first probe 202 and the second probe 204 may be prepared in the same reaction mixture and enabled to specifically interact with any first target 206 and second target 208, respectively, in the reaction mixture. As shown, the reaction mixture is subjected to at least two different reaction conditions. The first probe 202 is designed such that the first label 210 generates a first label signal 214 to an extent that correlates (e.g., is proportional) with the amount of specific interaction between the first probe 202 and the first target 206 in both the first set of conditions 218 and the second set of conditions 220. In contrast, the second probe 204 is designed such that the second label 212 generates a second label signal 216 to an extent that correlates (e.g., is proportional) with the amount of specific interaction between the second probe 204 and the second target 208 in the second set of conditions 220 rather than in the first set of conditions 218. In other words, under the first set of conditions 218, the first label signal 214 increases as a result of the specific interaction between the first probe 202 and the first target 206, while the second label signal 216 is not emitted (increased) as a result of the specific interaction between the second probe 204 and the second target 208. Under the second set of conditions 220, the second label signal 216 increases as a result of the specific interaction between the second probe 204 and the second target 208, and the first label signal 214 also further increases to at least some extent from the first set of conditions 218 or remains at a high level.

[0093] Under the first set of conditions 218, the second label 212 does not generate a "substantial signal (e.g., fluorescence)", and thus the second label signal 216 is not substantially different from the background (i.e., baseline) level of the luminescence signal (e.g., fluorescence) in the reaction mixture. That is, under the first set of conditions 218, there may be some non-zero level of signal generated by the second label 212, but the second label signal 216 typically remains below the threshold that separates the background signal from a meaningful signal. This threshold can vary according to the specific test protocol and the requirements of the application, as described above.

[0094] In at least some embodiments, when both the first target 206 and the second target 208 are present in the reaction mixture, the second labeled signal 216 between the first set of conditions 218 and the second set of conditions 220 is significantly more different than the first labeled signal 214 is different between the first set of conditions 218 and the second set of conditions 220. Thus, the first labeled signal 214 can be somewhat different between the first set of conditions 218 and the second set of conditions 220, but this difference is typically less than the difference in the second labeled signal 216 between the first set of conditions 218 and the second set of conditions 220.

[0095] In various embodiments of the present disclosure, the manner of response to different sets of conditions is utilized to be different between the first labeled signal 214 and the second labeled signal 216, such that the detected first label 214 and the second labeled signal 216 can be resolved (separated), even if, for example, they are detected within the same detection channel. As shown, the total signal (“first total signal”) is measured at the first set of conditions 218, and the signal (“second total signal”) is measured at the second set of conditions 220. The fluorescence (or luminescence) signal data representing the first total signal may be referred to herein as “first fluorescence signal data” or “first luminescence signal data”, and the fluorescence signal data representing the second total signal may be referred to herein as “second fluorescence signal data” or “complex fluorescence signal data” or “second luminescence signal data” or “complex luminescence signal data”. As used herein, first and second in this context are not necessarily used to indicate a temporal order of detection or conditions, although such a temporal order may occur.

[0096] In the first set of conditions 218, the total signal is substantially equal to the first labeled signal 214. That is, the first total signal consists mainly of the first labeled signal 214, but the contribution of the second labeled signal 216 can be ignored. In the second set of conditions 220, the total signal will include a combination of the first labeled signal 214 and the second labeled signal 216. Thus, the first labeled signal 214 and the second labeled signal 216 can be separately decomposed based on the first total signal and the second total signal. For example, the first labeled signal 214 can be determined based on the first total signal, and the second labeled signal 216 can be decomposed by subtracting the first total signal from the second total signal.

[0097] In some embodiments, the first labeled signal 214 is equal to the first total signal as is. In other embodiments, the first labeled signal 214 is determined as a function of the first total signal. In some embodiments, this function is a linear function (although in some embodiments, a non - linear function may be used). For example, as described above, the first labeled signal 214 may vary slightly between the first set of conditions 218 and the second set of conditions 220 even when the amount of the first target 206 has not changed. In a particular application, the first labeled signal 214 under the second set of conditions 220 may better conform to a standard curve where the first labeled signal 214 is equal to the amount of the first target 206. Thus, by estimating the first labeled signal 214 as a function of the first total signal rather than as being equal to the first total signal as is, the calculated first labeled signal 214 can be brought closer to that measured under the second set of conditions 220 (i.e., in the absence of the interfering second labeled signal 216).

[0098] In some embodiments, the function for converting the first total signal to the first labeled signal 214 is determined by comparing the first labeled signal 214 under the first set of conditions 218 in the absence of any second probe that interacts with the second target, with the first labeled signal 214 under the second set of conditions 220. The first labeled signal 214 under the first set of conditions 218 and the first labeled signal 214 under the second set of conditions 220 often correlate with each other according to a linear function. In other embodiments, a non-linear function can be used to correlate them. When using a linear function, a multiplier (e.g., a correction factor) can be used to convert the first total signal to the first labeled signal 214. Once such a linear function is determined, it can be used in subsequent assays without necessarily further comparing the first labeled signal 214 under the first set of conditions 218 and under the second set of conditions 220 in the absence of the second probe having the second target. In some embodiments, the function for converting the first total signal to the first labeled signal can be non-linear. In some embodiments, the function / correlation is determined over the stages of a thermal cycle where the number of cleaved probes is expected to be the same, or between thermal cycles. Using this approach, different signals can be resolved, for example, even when detected within the same detection channel.

[0099] As will be described in more detail below, the first probe 202 and the second probe 204 have different mechanisms of action that allow for different signal responses to the first set of conditions 218 and the second set of conditions 220, depending on the probe type. Advantageously, it is not necessary to rely on attributes such as different melting temperatures for the probes to enable the resolution of the distinct signals associated with each of the different probe types. Thus, the first probe 202 and the second probe 204 may have different melting temperatures, but that is not a requirement for effectively resolving their associated labeled signals. In some embodiments, for example, the melting temperature (T m)(Generally defined as the temperature at which 50% of the strands are in double-stranded form and 50% are single-stranded) and the T of the second probe 204 m are within about 8 °C, or about 6 °C, or about 4 °C or about 2 °C of each other. In some embodiments, under the first set of conditions or the second set of conditions, both probes bind (e.g., hybridize) to their respective targets. Optionally, under the first set of conditions or the second set of conditions, both probes do not substantially bind (e.g., hybridize) to their respective targets. In one example, under the first set of conditions, both the first probe and the second probe substantially bind (e.g., hybridize) to their respective targets, but under the second set of conditions, both the first probe and the second probe do not substantially bind (e.g., hybridize) to their respective targets. In another example, under the first set of conditions, both the first probe and the second probe do not necessarily substantially bind (e.g., hybridize) to their respective targets, but under the second set of conditions, both the first probe and the second probe substantially bind (e.g., hybridize) to their respective targets.

[0100] Figure 2B is a graph showing the signal response over time when the reaction mixture is cycled between a first set of reaction conditions 218 and a second set of reaction conditions 220 for the method outlined in Figure 2A, with both the first target 206 and the second target 208 present in the reaction mixture. Cycling of the conditions can include different conditions for various stages associated with thermal cycling in a nucleic acid amplification reaction, such as PCR. Under such reactions, the first set of reaction conditions 218 can correspond to assisting the denaturation stage, and the second set of reaction conditions 220 can correspond to assisting the annealing and / or extension stage (the "annealing / extension stage") of the thermal cycling. Thus, in various embodiments, the first set of reaction conditions 218 includes a first temperature or a first temperature range, and the second set of reaction conditions 220 includes a second temperature or a second temperature range (lower than the first).

[0101] As shown, under the second set of reaction conditions 220, both the first labeled signal 214 and the second labeled signal 216 increase. Under the first set of reaction conditions 218, the first labeled signal 214 remains substantially the same as at the end of the previous cycle (although it may vary slightly as described above), while the second labeled signal 216 decreases to a level equivalent to the baseline signal level of the second labeled signal 216, which may be substantially constant over a plurality of amplification cycles. In other words, the second labeled signal 216 exhibits a baseline signal above the background signal level under the first set of reaction conditions. In some cases, the second labeled signal may exhibit a baseline signal level that varies at different stages of the amplification cycle, but nevertheless is clearly distinguishable from and lower than the level under the second set of reaction conditions. This may be due to the different states of the probes and the proximity of the quencher and the label.

[0102] As shown, both the first labeled signal 214 and the second labeled signal 216 increase cumulatively each time the second set of conditions 220 occurs. This is the result of further specific interactions occurring between the first probe 202 and the first target 206 in the reaction mixture and between the second probe 204 and the second target 208 in the reaction mixture. However, when the first labeled signal 214 remains at an equivalent level when transitioning from the end of one cycle to the start of another cycle (i.e., when transitioning from the second set of conditions 220 at the end of a cycle to the first set of conditions 218 at the start of the next cycle), the second labeled signal 216 returns to a level near the baseline at the start of each cycle (i.e., at the point when the first set of conditions 218 occurs).

[0103] Some embodiments described herein can be utilized using in-channel multiplexing (detection within the same channel), but the present disclosure is not limited thereto. Further, in the present disclosure, inter-channel multiplexing and in-channel multiplexing combined with inter-channel multiplexing are also contemplated to further increase the multiplexity of the assay. For example, an assay may be designed using a plurality of different dyes (and thus using a plurality of different detection channels), and two or more of the different channels each contain a plurality of detectable signals that can be resolved using the techniques and methods described herein.

[0104] Cleavable probes and non-cleavable probes In some embodiments, the first probe (e.g., the first probe 202) is a "cleavable" probe. The first probe can be designed such that as a result of hybridization of the first probe with the first target (e.g., the first target 206), the first label (e.g., the first label 210) is separated from the first probe (and, e.g., released from a corresponding quencher). Thus, after being released, the first label continues to contribute to the total signal in the reaction mixture. The first probe can be, for example, a TaqMan probe that undergoes cleavage as a result of the 5'→3' exonuclease activity of DNA polymerase during the extension of the target molecule to which the probe hybridizes. TaqMan probes are described in U.S. Patent Nos. 4,889,818, 5,079,352, 5,210,015, 5,436,134, 5,487,972, 5,658,751, 5,210,015, 5,487,972, 5,538,848, 5,618,711, 5,677,152, 5,723,591, 5,773,258, 5,789,224, 5,801,155, 5,804,375, 5,876,930, 5,994,056, 6,030,787, 6,084,102, 6,127,155, 6,171,785, 6,214,979, 6,258,569, 6,814,934, 6,821,727, 7,141,377 and 7,445,900, all of which are incorporated herein by reference.

[0105] In some embodiments, the second probe (e.g., second probe 204) is a "non-cleavable" probe. The second label of the non-cleavable probe (e.g., second label 212) is intended to remain associated with the probe throughout the assay and for the level of the signal (e.g., second label signal 216) to change according to the structure of the probe rather than the release of the label. The second probe may be, for example, an extendable fluorogenic ("EF") probe, which quenches the label when in single-stranded form but allows the signal when incorporated into a double-stranded molecule.

[0106] In some embodiments, the second probe (e.g., second probe 204) is a compound or a salt thereof described later and a compound or a salt thereof described in the patent application entitled "Multiplex Dye Compounds" filed simultaneously with the United States Patent and Trademark Office on June 29, 2023, the entire content of which is incorporated herein by reference.

[0107] Figure 3A shows the activities of cleavable probe 302 (which may be a TaqMan probe in various embodiments) and non-cleavable probe 312 (which may be an EF probe in various embodiments) in the annealing, extension, and denaturation stages of the thermal cycle of a PCR reaction. As shown, in the annealing stage, TaqMan probe 302 hybridizes to its corresponding target nucleic acid amplicon 304 (as used herein, a target nucleic acid amplicon may refer to one strand of a target double-stranded nucleic acid and should be understood by reference to the context when describing the PCR reaction). During the process of primer 303 hybridizing to target nucleic acid amplicon 304 upstream of probe 302 and extending, the TaqMan probe label 306 is cleaved from the remainder of probe 302 by the 5’→3’ exonuclease activity of DNA polymerase, thereby separating from the corresponding TaqMan probe quencher 309. This results in a corresponding increase in the fluorescence signal. During the denaturation process, label 306 remains free in the reaction mixture solution and thus continues to contribute to the total fluorescence signal.

[0108] EF probe 312 includes EF probe label 316 and EF probe quencher 319, which remain in proximity to each other while probe 312 is in a single-stranded structure. Thus, the fluorescence signal from label 316 remains substantially quenched while the EF probe is in a single-stranded structure. In the annealing and extension stages, EF probe 312 hybridizes to its corresponding target template amplicon 314 and extends to form extended probe amplicon 313. Then, upon extension of target template 314, a complement 315 of extended probe amplicon 313 is formed. The resulting double-stranded amplicon 317 separates label 316 from quencher 319 to a distance sufficient to enable fluorescence emission. During the denaturation process, extended probe amplicon 313 separates from its complement 315. When it returns to the single-stranded structure, label 316 and quencher 318 return to the proximate state and fluorescence is quenched again.

[0109] Figure 3B is a graph showing the fluorescence signals over time from TaqMan probe 302 and EF probe 312 during the thermal cycling of the amplification process. The temperature of the thermal cycling can vary according to the needs of a particular application. As an example, the denaturation step can be carried out at an internal temperature in the range of about 80°C to about 100°C, such as about 85°C to about 95°C, or for example about 90°C to about 95°C. The annealing / extension step can be carried out at a lower temperature, such as in the range of about 40°C to about 75°C, for example about 50°C to about 70°C, for example about 55°C to about 65°C. In an embodiment, the first set of reaction conditions (e.g., the first set of conditions 218 described with reference to FIG. 2A) corresponds to the denaturation step 318, and the second set of reaction conditions (e.g., the second set of conditions 220 described with reference to FIG. 2A) corresponds to the annealing / extension step 320.

[0110] Various embodiments cycle between a denaturation step 318 and a combined annealing / extension step 320 (i.e., the amplification process cycles between two target temperatures), but other embodiments may include a separate annealing step and an extension step. In such embodiments, the temperature and possibly other reaction conditions can vary between the annealing step and the extension step. For example, the extension step can be carried out at a temperature higher than the annealing temperature. In some embodiments, the amplification process cycles between at least two target temperatures over at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cycles of the amplification process.

[0111] Figure 3B shows that the fluorescence signal associated with TaqMan probe 302 increases during the extension step 320 and then remains at a similar level through the denaturation step 318 of the next cycle, while the fluorescence signal associated with EF probe 304 increases during the extension step 320 but decreases to the baseline signal level associated with EF probe 304 when the subsequent denaturation step 318 reaches the target denaturation temperature. Those skilled in the art will understand that cycles N, N+1, and N+2 in Figure 3B can start from different stages, in which case the above comparison of signal levels may shift.

[0112] In some embodiments, the first set of reaction conditions (e.g., denaturation condition 318) includes a first measurement temperature at which the first labeled signal is measured, and the second set of reaction conditions (e.g., annealing / extension condition 320) includes a second different measurement temperature at which the first labeled signal and the second labeled signal are measured. In some embodiments, the first measurement temperature and the second measurement temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more. The first measurement temperature may be, for example, a target denaturation temperature in the range of about 80°C to about 100°C, for example, about 85°C to about 95°C, or for example, about 90°C to about 95°C, and the second measurement temperature may be, for example, a target annealing / extension temperature in the range of about 40°C to about 75°C, for example, about 50°C to about 70°C, for example, about 55°C to about 65°C.

[0113] Formation of EF Probe Template Figure 4A shows a process of using a primer (also referred to herein as a tailed primer) 422 having a tail specific to nucleic acid target 424 to form a target template 414 to which EF probe 412 can hybridize. The tailed primer 422 includes a tail 426 and a target-specific portion 428. Figure 4B shows a more detailed view of the tailed primer 422 as a forward primer, the target-specific primer 423 paired with the tailed primer 422 as a reverse primer, and the EF probe 412.

[0114] As shown, in the first step, the target - specific moiety 428 hybridizes to the target 424. The extension of the target - specific moiety 428 forms the tailed amplicon 425. The primer 423 paired with the tailed primer 422 enables the extension of the complement of the tailed amplicon 425. It is this complement that forms the target template 414. As shown, the target template 414 includes the tail - complementary portion 427.

[0115] In the second step, the EF probe 412 hybridizes to the target template 414 and, as shown in Figure 3A, amplification can continue. As shown in Figure 4A, the EF probe 412 includes a probe tail 417 that has substantial homology to the probe tail 426 and is thus complementary to the tail - complementary portion 427 of the target template 414. The extension of the probe 412 and the target template 414 forms the double - stranded amplicon 419. In the third step, the primer 423, shown paired here with the tailed primer 422, also functions as the primer 423 paired with the EF probe 412, as shown in Figure 3A, to enable the formation of the double - stranded amplicon 419.

[0116] As shown in Figure 4B, the tail 426 can form the 5'-end of the tailed primer 422. The EF probe 412 can include a stem - loop portion that, when the EF probe 412 is single - stranded, has stem portions 410 on both sides of the loop portion 411 and is configured to form a stem - loop structure. For example, the label 416 may be located on one side of the stem - loop portion and the quencher 418 may be located on the opposite side of the stem - loop portion, such that the label 416 and the quencher 418 are in proximity when the stem - loop structure is formed, but are more distantly arranged when the EF probe 412 is constrained to a more linear structure (e.g., when incorporated into a double - stranded amplicon).

[0117] In the embodiment shown, the label 416 is located at or near the 5'-end of the EF probe 412, and the quencher 418 is located on the 3'-side of the label 416. The positions of the label 416 and the quencher 418 may be reversed in other embodiments. Preferably, as shown, the stem-loop portion is arranged on the 5'-side of the probe tail 417 such that the stem-loop portion remains at the end of the amplicon resulting from the extension of the EF probe 412, so that (when single-stranded) the formation of the stem-loop structure is less likely to be impaired.

[0118] In some embodiments, the EF probe includes a non-stem-loop portion that separates a label (located at or near the 5'-end of the EF probe) from a quencher located at or near the 3'-end of the EF probe.

[0119] In addition to, or instead of, the EF probes described herein, some embodiments may include other labeled oligonucleotides that generate increased fluorescence (compared to when in single-stranded form) when incorporated into a double-stranded amplicon, for example, during the extension and / or annealing steps of a PCR process. For example, LUX™ primers include an internal fluorophore that is quenched by a hairpin structure located on the 5'-side of the fluorophore. Similar to the EF probe, the LUX™ primer is incorporated into the double-stranded amplicon and fluorescence increases when the hairpin structure becomes linear. Further, any of the primers or probes described herein may include one or more locked nucleic acids (LNAs) known in the art.

[0120] In some embodiments, a tailed primer 422 and a corresponding (non-tailed) primer 423 are prepared at different concentrations. For example, primer 423 may be prepared at a higher concentration than tailed primer 422. For example, primer 423 may be prepared at a concentration of about 2× (2 times) to about 30× (30 times) the concentration of tailed primer 422, or about 5× to about 25× the concentration of tailed primer 422, or about 10× to about 20× the concentration of tailed primer 422. Since primer 423 can function to drive both (1) the formation of the target template 414 (as shown in FIG. 4A) and (2) the formation of the complement 415 of the extension probe amplicon 413 (as shown in FIG. 3A), preparing it at a higher concentration than the corresponding tailed primer 422 can beneficially balance the reaction and increase the overall reaction efficiency.

[0121] In some embodiments, the EF probe 412 is prepared at a concentration different from the concentration of the tailed primer 422 and / or the concentration of the primer 423. For example, the EF probe 412 may be prepared at a concentration higher than the concentration of the tailed primer 422 and lower than the concentration of the primer 423. In some embodiments, the EF probe 412 is prepared at a concentration of about 2× to about 20× the concentration of the tailed primer 422, or about 3× to about 15× the concentration of the tailed primer 422. As described above, preparing the primer 423 at a relatively high concentration can increase the overall reaction efficiency. By preparing the EF probe 412 at a concentration higher than the tailed primer 422 but not necessarily higher than the primer 423, more related amplification is directed to the EF probe 412 as opposed to the tailed primer 422, and yet the primer 423 can still function as the main factor for increasing the reaction efficiency.

[0122] In addition to, or instead of, using the "general purpose" EF probe with the probe tail 417, other embodiments include and / or utilize an EF probe having a target-specific moiety instead of the probe tail 417. Such an EF probe can hybridize directly to the target template nucleic acid, as shown in FIG. 4A, and thus does not need to follow the two-step process shown in FIG. 4A to generate a target template 414 having a tail complementary moiety 427. In such embodiments, the probe tail 417 of the EF probe 412 is replaced with a target-specific moiety that hybridizes directly to the target 424. The process is otherwise substantially the same as that shown in FIG. 3A. That is, after the EF probe is extended, in the next annealing / extension round, the complementary strand is extended to form a double-stranded amplicon that separates the fluorophore and quencher, enabling fluorescence signal generation.

[0123] FIG. 4C shows a three-step thermal cycling method that can be used in an amplification process involving a non-cleavable probe (e.g., an EF probe) and optionally a cleavable probe (e.g., a TaqMan probe). The amplification process shown in FIG. 4C can be used in combination with any of the other methods disclosed herein. The amplification process shown includes a first step using a first target annealing / extension temperature, a second step using a second different annealing / extension temperature, and a third step using a third annealing / extension temperature. In this embodiment, the third annealing / extension temperature is the same as the first annealing / extension temperature. Other embodiments can include a third annealing / extension temperature that is different from both the first annealing / extension temperature and the second annealing / extension temperature.

[0124] Thus, the amplification process shown includes a denaturation temperature and a plurality of different annealing / elongation temperatures that vary throughout the amplification process. In such an amplification process, there is primarily an initial stage (stage 1) where the target template 414 is formed, an intermediate stage (stage 2) where the interaction between the EF probe 412 and the target template 414 increases to form the first extension probe amplicon 413, and a later stage (stage 3) where amplification can proceed further to include the probe amplicon 413 and the probe amplicon 415. This is beneficial in that there are these distinct stages.

[0125] As shown, the first annealing / elongation temperature may be higher than the second annealing / elongation temperature. The first series of denaturation steps and annealing / elongation steps (in stage 1) are cycled more times than the second series of denaturation steps and annealing / elongation steps (in stage 2). The third series of denaturation steps and annealing / elongation steps (in stage 3) can be cycled more times than the first series of denaturation steps and annealing / elongation steps. The denaturation temperature may be the same or different at each stage. As a non-limiting example, the denaturation temperatures at various stages can be above 80 °C but different from each other. For example, the temperature at an earlier stage can be higher than that at a later stage. In one embodiment, the denaturation temperature in the first stage can be about 95 °C and the denaturation temperature in the second stage can be about 85 °C.

[0126] Thus, stages 1 and 2 function as a pre-loading stage that, for at least one cycle, primarily generates the target template 414 (in stage 1) and then sets a lower annealing / elongation temperature to allow for an increase in the interaction between the EF probe 412 and the target template 414. Thereafter, a plurality of amplification cycles can be performed at the third annealing / elongation temperature to drive amplification that primarily includes the EF probe 412, the primer 423, and / or their extension probe amplicons 413 and 415. Thus, most of the amplification cycles are typically performed in stage 3.

[0127] The various specific temperatures, times, and temperature change rates shown in FIG. 4C are merely illustrative and should not be understood as limiting the scope of the present disclosure and claims. Other specific temperatures, times, and temperature change rates can be used, and these will be understood from the remainder of the present disclosure.

[0128] Embodiments are not limited to a three-step thermal cycling method. A one-step thermal cycling method (annealing / elongation temperature and denaturation temperature do not change for each cycle) or a two-step thermal cycling method (either the annealing / elongation temperature or the denaturation temperature changes for each cycle) can be used in an amplification process including a non-cleavable probe (e.g., an EF probe) and optionally a cleavable probe (e.g., a TaqMan probe).

[0129] Embodiments of digital PCR PCR is an in vitro method for enzymatically synthesizing a specific DNA sequence using two oligonucleotide primers that hybridize to opposite strands and are adjacent to a target region of the target DNA. Through a series of repetitive reaction steps including template denaturation, primer annealing, and elongation of the annealed primers by DNA polymerase, specific fragments defined by the primers at their ends accumulate exponentially. By PCR, a specific DNA sequence can be selectively concentrated by several orders of magnitude. As described above, the annealing step and the elongation step can be separate steps under different reaction conditions or can occur under the same series of reaction conditions.

[0130] Digital polymerase chain reaction (dPCR) is a specific application of PCR that can be used to directly quantify target nucleic acids in a sample. In dPCR, the reaction mixture is divided into a large number of small reaction volumes (also called fractions), such that the target nucleic acid is present in only some, and not all, of the reaction volumes. The reaction volumes are subjected to thermal cycling, and the proportion of "positive" reaction volumes that generate a signal indicating the presence of the target (e.g., a luminescent signal such as fluorescence from a detectable label) is determined. Quantification is based on the application of Poisson statistics, using the number of negative / non-reactive reaction volumes and assuming a Poisson distribution to determine the number of original copies that were distributed across the entire set of reaction volumes.

[0131] The features and principles described herein with respect to other amplification processes are generally applicable to the dPCR process as well. Accordingly, while the description in this section provides a more detailed disclosure of dPCR embodiments, the disclosure provided elsewhere in this document is also applicable to dPCR embodiments. In embodiments that include dPCR, various partitioning mechanisms or partitioning devices known in the art or that may be developed in the future may be utilized. For example, in some conventional dPCR systems, a plurality of droplets encapsulated by an oil phase are utilized to form a plurality of fractions / reaction volumes. In other embodiments, an array of microchambers may be utilized. An example of such a system is the QuantStudio Absolute Q system available from Thermo Fisher Scientific, which uses a microfluidic array plate to perform sample partitioning / division and the creation of reaction volumes. One of ordinary skill in the art is familiar with the various types of systems for partitioning a sample into small reaction volumes, subjecting those reaction volumes to PCR, and detecting the luminescent signal from the reaction volumes.

[0132] In some dPCR embodiments, the reaction mixture is fully formed before being dispensed into a plurality of reaction volumes. In alternative embodiments, one or more components of the reaction mixture may be added onto or into the reaction volume. For example, a probe and / or primer may be coated onto the walls of the microchamber, and then a sample and / or other components of the reaction mixture are added to the microchamber to form a plurality of reaction mixtures in each reaction volume.

[0133] Figure 5A is a schematic diagram of a method for detecting a plurality of target nucleic acids using a PCR process and a probe carrying a detectable label having spectral similarity. The PCR process can typically include real-time / quantitative PCR (qPCR) that monitors amplification during the reaction, and dPCR typically includes endpoint measurements and / or endpoint PCR for determining by counting the number of "positive" fractions. Thus, Figure 5A shows that under both a first set of conditions 518 (e.g., denaturation conditions such as about 95 °C) and a second set of conditions 520 (e.g., annealing / extension conditions such as about 65 °C), a first probe 502 configured to associate with a first label 510 and specifically interact with a first target 506 is shown as positive (+) at the end of the reaction because there is emission from the first label signal 514. On the other hand, a second probe 504 associated with a second label 512 and configured to specifically interact with a second target 508 is shown as negative (-) under the first set of conditions 518 because there is no emission signal from the second label signal 516, but is shown as positive (+) under the second set of conditions 520 because there is an emission signal from the second label signal 516.

[0134] This is similar to the scheme shown in Figure 2A, and Figure 5A shows that the same principle can be applied to endpoint measurements such as dPCR. As described in other embodiments, the first probe 502 can be a cleavable probe such as a TaqMan probe, and the second probe 504 can be a non-cleavable probe such as an EF probe.

[0135] Figure 5B shows how the signal (e.g., at the endpoint cycle of PCR) regarding the dPCR reaction volume can vary depending on whether the first probe 502, the second probe 504, or both were active within the reaction volume during the reaction. As shown, when the signal 514 is generated only from the first probe 502, the reaction volume is positive (+) under both the first set of conditions 518 and the second set of conditions 520. When the signal 516 is generated only from the second probe 504, the reaction volume is negative (-) under the first set of conditions 518 and positive (+) under the second set of conditions 520. When signals are generated from both the first probe 502 and the second probe 504, the reaction volume is positive (+) under the first set of conditions 518 and strongly positive (++) (a strong luminescence signal resulting from the contributions of both the labeled signal 514 and the labeled signal 516) under the second set of conditions 520. Naturally, when no signal is generated from either probe type, the reaction volume is negative (-) under any set of conditions 518, 520.

[0136] The total number of reaction volumes that are positive for the first probe 502 (and thus presumed to be positive for the first target 506) is determined by counting the number of reaction volumes that are positive (+) or strongly positive (++) under both sets of conditions 518, 520. The total number of reaction volumes that are positive for the second probe 504 (and thus presumed to be positive for the second target 508) is determined by (i) counting the number of reaction volumes that are positive (+) under the second set of conditions 520 but negative (-) under the first set of conditions 518, and adding that to (ii) the number of reaction volumes that are strongly positive (++) under the second set of conditions 520. These numbers of reaction volumes can be calculated or estimated by plotting the signal under the first set of conditions 518 at the endpoint cycle of PCR against the signal under the second set of conditions 520 at the endpoint cycle and identifying clusters. See, for example, the plot of FIG. 8 described in more detail in the Examples section below. The concentrations of the first target 506 and the second target 508 in the sample can then be estimated using standard dPCR techniques.

[0137] Accordingly, a method for determining the presence and / or amount of multiple targets using the multiplexing techniques described herein for dPCR applications may include preparing a reaction mixture comprising a first nucleic acid target and a first probe type (e.g., TaqMan probe) and a second probe type (e.g., EF probe) designed to specifically interact with the second nucleic acid target, respectively; adding / allocating the sample into a plurality of reaction volumes; measuring the signal of the reaction volume under a first set of reaction conditions (e.g., denaturation conditions such as about 95° C.) during the endpoint cycle of PCR; measuring the signal of the reaction volume under a second set of reaction conditions (e.g., annealing / extension conditions such as about 65° C.) during the endpoint cycle of PCR; classifying the reaction volumes according to the signal characteristics measured by the endpoint signal measurement; determining or estimating the number for each probe type (i.e., the number of reaction volumes in which the first probe type was active and the number of reaction volumes in which the second probe type was active) based on the classified reaction volumes; and determining or estimating the presence and / or amount of the first nucleic acid target and the second nucleic acid target based on those numbers.

[0138] Embodiments of endpoint PCR The analysis techniques according to various embodiments can be used in the same manner as is well known to those skilled in the art when performing a conventional endpoint PCR process that subjects a sample (or in a larger reaction volume that does not aim to capture a single DNA molecule using Poisson statistics or to capture it) to PCR in large quantities. In such a PCR process, the measurement of signals from two different probe types can be carried out in the endpoint cycle of PCR and under different reaction conditions (such as the denaturation conditions and annealing and / or extension conditions described herein) in the same manner as the approach described above for the dPCR process. However, the detected labeled signals will follow those schematically shown in Figure 2A, and thus the signals will indicate the presence or absence of the first target and the second target, respectively. Thus, referring to Figure 3B, the endpoint cycle signals measured under two different reaction conditions can result in different levels of signals (analogous to that in the endpoint cycle) schematically shown at cycle N+2. By comparing the different signal levels obtained using the algorithm shown in Figure 2A and further described above, it is possible to determine the presence or absence of the first target nucleic acid and the second target nucleic acid using detectable labels that have overlapping emission signal spectra and are detectable in the same detection channel.

[0139] Accordingly, the method for determining the presence or absence of a plurality of targets using the features of in-channel multiplexing described herein can also be carried out using endpoint PCR (i.e., using a large or larger reaction volume that is not sized to rely on Poisson statistics, as is well known to those skilled in the art, to capture single molecules of target nucleic acid in the reaction volume). The method used in endpoint PCR applications comprises preparing a reaction mixture comprising a first probe type (e.g., TaqMan probe) configured to specifically interact with a first target nucleic acid and a second nucleic acid target, respectively, and a second probe type (e.g., EF probe); subjecting the reaction mixture to an amplification reaction (e.g., PCR); measuring the endpoint cycle signal of the reaction mixture under a first set of reaction conditions (e.g., denaturation conditions such as about 95° C.); measuring the endpoint signal of the reaction volume under a second set of reaction conditions (e.g., annealing / extension conditions such as about 65° C.) (in particular, for measurements at the endpoint, the measurement under the second set of reaction conditions (annealing / extension) is carried out prior to the measurement under the first set of reaction conditions (denaturation)); measuring a first total emission (e.g., fluorescence) signal comprising any first emission (e.g., fluorescence) signal, if present, under the first set of reaction conditions; measuring a second total emission (e.g., fluorescence) signal comprising any first emission (e.g., fluorescence) signal, if present, and any second emission (e.g., fluorescence) signal, if present, under the second set of reaction conditions; and determining the presence or absence of the first nucleic acid target and / or the second nucleic acid target in the reaction mixture by estimating the first emission (e.g., fluorescence) signal and / or the second emission (e.g., fluorescence) signal based on the first total emission (e.g., fluorescence) signal and the second total emission (e.g., fluorescence) signal.

[0140] Compound In one embodiment, the second probe or non-cleavable probe has the formula:

[0141] [Chemical formula] A compound having or a salt thereof, wherein Q A is a quencher moiety, B is a divalent nucleobase, L 1 is a divalent linker, R 2 is hydrogen or -OR 2A wherein, R 3 is -OR 3A or -O-P(NR 3B R 3C )-OR 3A wherein, R 4 is hydrogen or unsubstituted methyl, or the R 2 substituent and the R 4 substituent combine to form a substituted or unsubstituted heterocycloalkyl (e.g., 3- to 8-membered, 3- to 6-membered, 4- to 6-membered, 4- to 5-membered or 5- to 6-membered), R 5 is -OR 5A and R 2A 、R 3A 、R 3B 、R 3C and R 5A are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, a substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), a substituted or unsubstituted heteroalkyl (e.g., 2- to 8-membered, 2- to 6-membered, 4- to 6-membered, 2- to 3-membered or 4- to 5-membered), a substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), a substituted or unsubstituted heterocycloalkyl (e.g., 3- to 8-membered, 3- to 6-membered, 4- to 6-membered, 4- to 5-membered or 5- to 6-membered), a substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0142] In an embodiment, the quencher moiety is the monovalent form of QSY7.

[0143] In an embodiment, the quencher moiety is the monovalent form of

[0144]

Chemical Structure

[0145] In an embodiment, the quencher moiety is

[0146]

Chemical Structure

[0147] In an embodiment, the quencher moiety is the monovalent form of QSY21.

[0148] In an embodiment, the quencher moiety is the monovalent form of

[0149]

Chemical Structure

[0150] In an embodiment, the quencher moiety is

[0151]

Chemical Structure

[0152] In an embodiment, the quencher moiety is the monovalent form of QSY9.

[0153] In the embodiment, the quencher moiety is in the monovalent form of

[0154] [Chemical Formula] and is as follows.

[0155] In the embodiment, the quencher moiety is

[0156] [Chemical Formula] and is as follows.

[0157] In the embodiment, the quencher moiety is the monovalent form of BHQ1.

[0158] In the embodiment, the quencher moiety is in the monovalent form of

[0159] [Chemical Formula] and is as follows.

[0160] In the embodiment, the quencher moiety is

[0161] [Chemical Formula] and is as follows.

[0162] In the embodiment, the quencher moiety is the monovalent form of BHQ2.

[0163] In the embodiment, the quencher moiety is in the monovalent form of

[0164] [Chemical Formula] and is as follows.

[0165] In the embodiment, the quencher part is

[0166]

Chemical formula

[0167] In the embodiment, the quencher part is the monovalent form of BHQ3.

[0168] In the embodiment, the quencher part is the monovalent form of

[0169]

Chemical formula

[0170] In the embodiment, the quencher part is

[0171]

Chemical formula

[0172] In the embodiment, the quencher part is the monovalent form of Dabcyl.

[0173] In the embodiment, the quencher part is the monovalent form of

[0174]

Chemical formula

[0175] In the embodiment, the quencher part is

[0176]

Chemical formula

[0177] In the embodiment, the quencher moiety is the monovalent form of Dabsyl.

[0178] In the embodiment, the quencher moiety is the monovalent form of

[0179]

Chemical formula

[0180] In the embodiment, the quencher moiety is the monovalent form of Eclipse.

[0181] In the embodiment, the quencher moiety is the monovalent form of

[0182]

Chemical formula

[0183] In the embodiment, the quencher moiety is

[0184]

Chemical formula

[0185] In the embodiment, the quencher moiety is the monovalent form of BBQ-650.

[0186] In the embodiment, the quencher moiety is

[0187]

Chemical formula

[0188] In the embodiment, the quencher moiety is the monovalent form of Iowa Black RQ.

[0189] In an embodiment, the quencher moiety is the monovalent form of Iowa Black FQ.

[0190] In an embodiment, all of the above quencher moieties are interchangeable. In an embodiment, the quencher moiety can be replaced in the formulas (I), (IA), (II), (III), (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (VI-4), (VI-5), (VII), (VII-1), (VII-2), (VII-3), (VII-4), (VII-5), (VIII), (VIII-1), (VIII-2), (VIII-3), (VIII-4), (VIII-5), (IX), (IX-1), (IX-2), (IX-3), (IX-4), (IX-5), (X), (XI), (XII), (XIII), (XIV), (XV), (XV-1), (XV-2), (XV-3), (XV-4), (XV-5), (XVI), (XVI-1), (XVI-2), (XVI-3), (XVI-4), (XVI-5), (XVII), (XVII-1), (XVII-2), (XVII-3), (XVII-4), (XVII-5), (XVIII), (XVIII-1), (XVIII-2), (XVIII-3), (XVIII-4), (XVIII-5) and (XIX), and in their embodiments.

[0191] In an embodiment, the second probe or non-cleavable probe is the following formula:

[0192]

Chemical formula

[0193] In an embodiment, R 1 and R 6 are bonded to form a substituted or unsubstituted heterocycloalkyl (e.g., 3- to 8-membered, 3- to 6-membered, 4- to 6-membered, 4- to 5-membered or 5- to 6-membered), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered or 5- to 6-membered).

[0194] In an embodiment, R 8 and R 10 are bonded to form a substituted or unsubstituted heterocycloalkyl (e.g., 3- to 8-membered, 3- to 6-membered, 4- to 6-membered, 4- to 5-membered or 5- to 6-membered), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered or 5- to 6-membered).

[0195] R 2A , R 3A , R 3B , R 3C , R 5A and R Ais, independently, hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2- to 8-membered, 2- to 6-membered, 4- to 6-membered, 2- to 3-membered or 4- to 5-membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3- to 8-membered, 3- to 6-membered, 4- to 6-membered, 4- to 5-membered or 5- to 6-membered), substituted or unsubstituted aryl (e.g., C6-C 10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered or 5- to 6-membered).

[0196] One of ordinary skill in the art will appreciate that the compound may exist as a neutral species having a counterion.

[0197] In an embodiment, the compound has the formula:

[0198]

Chemical formula

[0199] In an embodiment, the compound has the following formula:

[0200] [Chemical formula] and has the following groups: B, L 1 , R 2 , R 3 and R 5 are as described herein, including in the embodiments.

[0201] In an embodiment, the compound has the following formula:

[0202] [Chemical formula] and has the following groups: B, L 1 , R 2 , R 3 and R 5 are as described herein, including in the embodiments.

[0203] In an embodiment, the compound has the following formula:

[0204] [Chemical formula] and has the following groups:

[0205] B, L 1 , R 3 and R 5 are as described herein, including in the embodiments.

[0206] In an embodiment, the compound has the following formula:

[0207] [Chemical formula] and has the following groups: B, L 1 , R 3and R 5 is as described herein, including embodiments.

[0208] In an embodiment, B is a divalent cytosine or its derivative, divalent guanine or its derivative, divalent adenine or its derivative, divalent thymine or its derivative, divalent uracil or its derivative, divalent hypoxanthine or its derivative, divalent xanthine or its derivative, divalent 7-methylguanine or its derivative, divalent 5,6-dihydrouracil or its derivative, divalent 5-methylcytosine or its derivative, or divalent 5-hydroxymethylcytosine or its derivative. In an embodiment, B is a divalent cytosine or its derivative, divalent guanine or its derivative, divalent adenine or its derivative, divalent thymine or its derivative, or divalent uracil or its derivative. In an embodiment, B is a divalent cytosine or its derivative. In an embodiment, B is a divalent guanine or its derivative. In an embodiment, B is a divalent adenine or its derivative. In an embodiment, B is a divalent thymine or its derivative. In an embodiment, B is a divalent uracil or its derivative.

[0209] In an embodiment, the compound has the following formula:

[0210]

Chemical formula

[0211] In an embodiment, the compound has the following formula:

[0212]

Chemical formula

[0213] In an embodiment, the compound has the formula:

[0214]

Chemical formula

[0215] In an embodiment, the compound has the formula:

[0216]

Chemical formula

[0217] In an embodiment, the compound has the formula:

[0218]

Chemical formula

[0219] In an embodiment, the compound has the formula:

[0220]

Chemical formula

[0221] In an embodiment, the compound has the formula:

[0222]

Chemical formula

[0223] In an embodiment, the compound has the formula:

[0224]

Chemical formula

[0225] In an embodiment, the compound has the formula:

[0226]

Chemical formula

[0227] In an embodiment, the compound has the formula:

[0228]

Chemical formula

[0229] In an embodiment, the compound has the formula:

[0230]

Chemical formula

[0231] In an embodiment, the compound has the formula:

[0232]

Chemical formula

[0233] In an embodiment, the compound has the formula:

[0234]

Chemical formula

[0235] In an embodiment, the compound has the formula:

[0236]

Chemical formula

[0237] In an embodiment, the compound has the formula:

[0238]

Chemical formula

[0239] In an embodiment, the compound has the formula:

[0240]

Chemical formula

[0241] In an embodiment, the compound has the formula:

[0242]

Chemical formula

[0243] In an embodiment, the compound has the formula:

[0244]

Chemical formula

[0245] In an embodiment, the compound has the formula:

[0246]

Chemical formula

[0247] In an embodiment, the compound has the formula:

[0248]

Chemical formula

[0249] In an embodiment, the compound has the formula:

[0250]

Chemical formula

[0251] In an embodiment, the compound has the formula:

[0252]

Chemical formula

[0253] In an embodiment, the compound has the following formula:

[0254]

Chemical formula

[0255] In an embodiment, the compound has the following formula:

[0256]

Chemical formula

[0257] In an embodiment, L 1 is a divalent linker containing 4 to 30 atoms.

[0258] In an embodiment, L 1 is L 101 -L 102 -L 103 -L 104 -L 105 as follows.

[0259] L 101 , L 102 , L 103 , L 104 and L 105is independently a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkylene (e.g., 2- to 8-membered, 2- to 6-membered, 4- to 6-membered, 2- to 3-membered or 4- to 5-membered), substituted or unsubstituted cycloalkylene (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkylene (e.g., 3- to 8-membered, 3- to 6-membered, 4- to 6-membered, 4- to 5-membered or 5- to 6-membered), substituted or unsubstituted arylene (e.g., C6-C 10 or phenylene), or substituted or unsubstituted heteroarylene (e.g., 5- to 10-membered, 5- to 9-membered or 5- to 6-membered).

[0260] In embodiments, substituted L 101 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, a size-restricted substituent or a lower substituent, and when substituted L 101 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents, the substituents, size-restricted substituents and / or lower substituents may each optionally be different. In a plurality of embodiments, when L 101 is substituted, it is substituted with at least one substituent. In embodiments, when L 101 is substituted, it is substituted with at least one size-restricted substituent. In embodiments, when L 101 is substituted, it is substituted with at least one lower substituent.

[0261] In embodiments, L 101 is a bond. In embodiments, L 101 is -NH-. In embodiments, L 101 is -O-. In embodiments, L101 is -S-. In an embodiment, L 101 is -S(O)-. In an embodiment, L 101 is -S(O)2-. In an embodiment, L 101 is -C(O)-. In an embodiment, L 101 is -C(O)NH-. In an embodiment, L 101 is -NHC(O)-. In an embodiment L 101 is -NHC(O)NH-. In an embodiment, L 101 is -C(O)O-. In an embodiment, L 101 is -OC(O)-. In an embodiment, L 101 is substituted or unsubstituted C1-C4 alkylene. In an embodiment, L 101 is substituted or unsubstituted 2- to 6-membered heteroalkylene.

[0262] In an embodiment, substituted L 102 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent or lower substituent, and when substituted L 102 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents, the substituents, size-restricted substituents and / or lower substituents may each optionally be different. In an embodiment, L 102 when substituted, it is substituted with at least one substituent. In an embodiment, L 102 when substituted, it is substituted with at least one size-restricted substituent. In an embodiment, L 102 when substituted, it is substituted with at least one lower substituent.

[0263] In an embodiment, L 102 is a bond. In an embodiment, L 102 is -NH-. In an embodiment, L 102 is -O-. In an embodiment, L 102is -S-. In an embodiment, L 102 is -S(O)-. In an embodiment, L 102 is -S(O)2-. In an embodiment, L 102 is -C(O)-. In an embodiment, L 102 is -C(O)NH-. In an embodiment, L 102 is -NHC(O)-. In an embodiment, L 102 is -NHC(O)NH-. In an embodiment, L 102 is -C(O)O-. In an embodiment, L 102 is -OC(O)-. In an embodiment, L 102 is substituted or unsubstituted C1-C4 alkylene. In an embodiment, L 102 is substituted or unsubstituted 2-6 membered heteroalkylene. In an embodiment, L 102 is unsubstituted 3-8 membered heterocycloalkyl. In an embodiment, L 102 is unsubstituted piperidinyl. In an embodiment, L 102 is

[0264]

Chemical formula

[0265] In an embodiment, substituted L 103 (for example, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, a substituent with a restricted size or a lower substituent, and when substituted L 103 is substituted with a plurality of groups selected from substituents, substituents with a restricted size and lower substituents, the substituents, substituents with a restricted size and / or lower substituents may each optionally be different. In an embodiment, L 103 when substituted, it is substituted with at least one substituent. In an embodiment, L 103 when substituted, it is substituted with at least one substituent with a restricted size. In an embodiment, L103 When it is substituted, it is substituted with at least one lower substituent.

[0266] In an embodiment, L 103 is a bond. In an embodiment, L 103 is -NH-. In an embodiment, L 103 is -O-. In an embodiment, L 103 is -S-. In an embodiment, L 103 is -S(O)-. In an embodiment, L 103 is -S(O)2-. In an embodiment, L 103 is -C(O)-. In an embodiment, L 103 is -C(O)NH-. In an embodiment, L 103 is -NHC(O)-. In an embodiment, L 103 is -NHC(O)NH-. In an embodiment, L 103 is -C(O)O-. In an embodiment, L 103 is -OC(O)-. In an embodiment, L 103 is substituted or unsubstituted C1-C4 alkylene. In an embodiment, L 103 is substituted or unsubstituted 2- to 6-membered heteroalkylene.

[0267] In an embodiment, substituted L 104 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, a size-restricted substituent or a lower substituent, and when substituted L 104 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents, the substituents, size-restricted substituents and / or lower substituents may each optionally be different. In an embodiment, when L 104 is substituted, it is substituted with at least one substituent. In an embodiment, when L 104 is substituted, it is substituted with at least one size-restricted substituent. In an embodiment, L 104When it is replaced, it is substituted with at least one lower substituent.

[0268] In an embodiment, L 104 is a bond. In an embodiment, L 104 is -NH-. In an embodiment, L 104 is -O-. In an embodiment, L 104 is -S-. In an embodiment, L 104 is -S(O)-. In an embodiment, L 104 is -S(O)2-. In an embodiment, L 104 is -C(O)-. In an embodiment, L 104 is -C(O)NH-. In an embodiment, L 104 is -NHC(O)-. In an embodiment, L 104 is -NHC(O)NH-. In an embodiment, L 104 is -C(O)O-. In an embodiment, L 104 is -OC(O)-. In an embodiment, L 104 is unsubstituted C1-C 10 alkylene. In an embodiment, L 104 is unsubstituted methylene. In an embodiment, L 104 is unsubstituted ethylene. In an embodiment, L 104 is unsubstituted propylene. In an embodiment, L 104 is unsubstituted n-propylene. In an embodiment, L 104 is unsubstituted butylene. In an embodiment, L 104 is unsubstituted n-butylene. In an embodiment, L 104 is unsubstituted pentylene. In an embodiment, L 104 is unsubstituted n-pentylene. In an embodiment, L 104 is unsubstituted hexylene. In an embodiment, L 104 is unsubstituted n-hexylene. In an embodiment, L 104 is unsubstituted heptylene. In an embodiment, L 104 is unsubstituted n-heptylene. In an embodiment, L 104 is unsubstituted octylene. In an embodiment, L 104is unsubstituted n-octylene. In an embodiment, L 104 is unsubstituted C2-C6 alkynylene. In an embodiment, L 104 is unsubstituted ethynylene. In an embodiment, L 104 is unsubstituted propynylene. In an embodiment, L 104 is unsubstituted butynylene. In an embodiment, L 104 is unsubstituted pentynylene. In an embodiment, L 104 is unsubstituted hexynylene. In an embodiment, L 104 is

[0269]

Chemical formula

[0270]

Chemical formula

[0271]

Chemical formula

[0272] [Chem.] as follows.

[0273] In an embodiment, the substituent L 105 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent, a substituent with a limited size, or a lower substituent, and when the substituent L 105 is substituted with a plurality of groups selected from substituents, substituents with a limited size, and lower substituents, the substituents, substituents with a limited size, and / or lower substituents may each optionally be different. In an embodiment, when L 105 is substituted, it is substituted with at least one substituent. In an embodiment, when L 105 is substituted, it is substituted with at least one substituent with a limited size. In an embodiment, when L 105 is substituted, it is substituted with at least one lower substituent.

[0274] In an embodiment, L 105 is a bond. In an embodiment, L 105 is -NH-. In an embodiment, L 105 is -O-. In an embodiment, L 105 is -S-. In an embodiment, L 105 is -S(O)-. In an embodiment, L 105 is -S(O)2-. In an embodiment, L 105 is -C(O)-. In an embodiment, L 105 is -C(O)NH-. In an embodiment, L 105 is -NHC(O)-. In an embodiment, L 105 is -NHC(O)NH-. In an embodiment, L 105 is -C(O)O-. In an embodiment, L 105is -OC(O)-. In an embodiment, L 105 is unsubstituted C1 - C 10 alkylene. In an embodiment, L 105 is substituted or unsubstituted C1 - C4 alkylene. In an embodiment, L 105 is unsubstituted methylene. In an embodiment, L 105 is unsubstituted ethylene. In an embodiment, L 105 is unsubstituted propylene. In an embodiment, L 105 is unsubstituted n - propylene. In an embodiment, L 105 is unsubstituted butylene. In an embodiment, L 105 is unsubstituted n - butylene. In an embodiment, L 105 is unsubstituted pentylene. In an embodiment, L 105 is unsubstituted n - pentylene. In an embodiment, L 105 is unsubstituted hexylene. In an embodiment, L 105 is unsubstituted n - hexylene. In an embodiment, L 105 is unsubstituted heptylene. In an embodiment, L 105 is unsubstituted n - heptylene. In an embodiment, L 105 is unsubstituted octylene. In an embodiment, L 105 is unsubstituted n - octylene. In an embodiment, L 105 is unsubstituted C2 - C6 alkynylene. In an embodiment, L 105 is unsubstituted ethynylene. In an embodiment, L 105 is unsubstituted propynylene. In an embodiment, L 105 is unsubstituted butynylene. In an embodiment, L 105 is unsubstituted pentynylene. In an embodiment, L 105 is unsubstituted hexynylene. In an embodiment, L 105 is

[0275]

Chemical formula

[0276] [Chemical formula] In an embodiment, L 105 is

[0277] [Chemical formula] In an embodiment, L 105 is

[0278] [Chemical formula] In an embodiment, L 105 is a substituted or unsubstituted 2- to 8-membered heteroalkynylene. In an embodiment, L 105 is

[0279] [Chemical formula] In an embodiment, L 105 is

[0280] [Chemical formula] where n105 is an integer from 1 to 10. In an embodiment, n105 is 1. In an embodiment, n105 is 2. In an embodiment, n105 is 3. In an embodiment, n105 is 4. In an embodiment, n105 is 5. In an embodiment, n105 is 6. In an embodiment, n105 is 7. In an embodiment, n105 is 8. In an embodiment, n105 is 9. In an embodiment, n105 is 10. In an embodiment, L 105 is an unsubstituted 5- to 10-membered heteroarylene. In an embodiment, L 105 is an unsubstituted triazolylene. In an embodiment, L 105 is

[0281] [Chemical Formula] as follows.

[0282] In an embodiment, L 1 is

[0283] [Chemical Formula] as follows. In an embodiment, L 1 is

[0284] [Chemical Formula] as follows.

[0285] In an embodiment, L 1 is

[0286] [Chemical Formula] as follows. In an embodiment, L 1 is

[0287] [Chemical Formula] is. In an embodiment, L 1 is

[0288]

Chem.

[0289]

Chem.

[0290]

Chem.

[0291]

Chem.

[0292]

Chem.

[0293]

Chem.

[0294] In an embodiment, the substituent R 1 (for example, substituted alkyl and / or substituted heteroalkyl) is substituted with at least one substituent, a size-limited substituent or a lower substituent, and the substituent R 1When it is substituted with a plurality of groups selected from substituents, substituents with limited size, and lower substituents, the substituents, substituents with limited size, and / or lower substituents may each optionally be different. In an embodiment, R 1 when substituted, is substituted with at least one substituent. In an embodiment, R 1 when substituted, is substituted with at least one substituent with limited size. In an embodiment, R 1 when substituted, is substituted with at least one lower substituent.

[0295] In an embodiment, R 1 is hydrogen. In an embodiment, R 1 is unsubstituted C1-C4 alkyl. In an embodiment, R 1 is unsubstituted methyl. In an embodiment, R 1 is unsubstituted ethyl. In an embodiment, R 1 is unsubstituted propyl. In an embodiment, R 1 is unsubstituted n-propyl. In an embodiment, R 1 is unsubstituted isopropyl. In an embodiment, R 1 is unsubstituted butyl. In an embodiment, R 1 is unsubstituted n-butyl. In an embodiment, R 1 is unsubstituted isobutyl. In an embodiment, R 1 is unsubstituted tert-butyl.

[0296] In an embodiment, R 2 is hydrogen or -OH. In an embodiment, R 2 is hydrogen. In an embodiment, R 2 is -OR 2A . In an embodiment, R 2 is -OH.

[0297] In an embodiment, substituted R 2A(For example, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, a substituent with a limited size or a lower substituent, and substituted R 2A When is substituted with a plurality of groups selected from substituents, substituents with a limited size and lower substituents, the substituents, substituents with a limited size and / or lower substituents may each optionally be different. In embodiments, R 2A When is substituted, it is substituted with at least one substituent. In embodiments, R 2A When is substituted, it is substituted with at least one substituent with a limited size. In embodiments, R 2A When is substituted, it is substituted with at least one lower substituent.

[0298] In embodiments, R 2A is hydrogen. In embodiments, R 2A is unsubstituted C1-C4 alkyl. In embodiments, R 2A is unsubstituted methyl. In embodiments, R 2A is unsubstituted ethyl. In embodiments, R 2A is unsubstituted propyl. In embodiments, R 2A is unsubstituted n-propyl. In embodiments, R 2A is unsubstituted isopropyl. In embodiments, R 2A is unsubstituted butyl. In embodiments, R 2A is unsubstituted n-butyl. In embodiments, R 2A is unsubstituted isobutyl. In embodiments, R 2A is unsubstituted tert-butyl.

[0299] In embodiments, R 3 is -OR 3A . In embodiments, R 3 is -OH. In embodiments, R 3 is, -O-P(NR 3B R 3C )-OR3A It is. In an embodiment, R 3 is

[0300] [Chemical formula] It is.

[0301] In an embodiment, substituted R 3A (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent, a substituent with a limited size, or a lower substituent, and when substituted R 3A is substituted with a plurality of groups selected from substituents, substituents with a limited size, and lower substituents, the substituents, substituents with a limited size, and / or lower substituents may each optionally be different. In an embodiment, when R 3A is substituted, it is substituted with at least one substituent. In an embodiment, when R 3A is substituted, it is substituted with at least one substituent with a limited size. In an embodiment, when R 3A is substituted, it is substituted with at least one lower substituent.

[0302] In an embodiment, R 3A is hydrogen. In an embodiment, R 3A is unsubstituted C1-C4 alkyl. In an embodiment, R 3A is unsubstituted methyl. In an embodiment, R 3A is unsubstituted ethyl. In an embodiment, R 3A is unsubstituted propyl. In an embodiment, R 3A is unsubstituted n-propyl. In an embodiment, R 3A is unsubstituted isopropyl. In an embodiment, R 3A is unsubstituted butyl. In an embodiment, R 3A is unsubstituted n-butyl. In an embodiment, R 3Ais unsubstituted isobutyl. In an embodiment, R 3A is unsubstituted tert-butyl. In an embodiment, R 3A is substituted C1-C4 alkyl. In an embodiment, R 3A is cyano-substituted C1-C4 alkyl. In an embodiment, R 3A is

[0303] [Chemical formula] is as follows.

[0304] In an embodiment, substituted R 3B (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, a size-restricted substituent or a lower substituent, and when substituted R 3B is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents, the substituents, size-restricted substituents and / or lower substituents may each optionally be different. In an embodiment, when R 3B is substituted, it is substituted with at least one substituent. In an embodiment, when R 3B is substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when R 3B is substituted, it is substituted with at least one lower substituent.

[0305] In an embodiment, R 3B is hydrogen. In an embodiment, R 3B is unsubstituted C1-C4 alkyl. In an embodiment, R 3B is unsubstituted methyl. In an embodiment, R 3B is unsubstituted ethyl. In an embodiment, R 3B is unsubstituted propyl. In an embodiment, R 3B is unsubstituted n-propyl. In an embodiment, R 3Bis unsubstituted isopropyl. In an embodiment, R 3B is unsubstituted butyl. In an embodiment, R 3B is unsubstituted n-butyl. In an embodiment, R 3B is unsubstituted isobutyl. In an embodiment, R 3B is unsubstituted tert-butyl.

[0306] In an embodiment, substituted R 3C (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent, a size-restricted substituent, or a lower substituent, and when substituted R 3C is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents, the substituents, size-restricted substituents, and / or lower substituents may each optionally be different. In an embodiment, when R 3C is substituted, it is substituted with at least one substituent. In an embodiment, when R 3C is substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when R 3C is substituted, it is substituted with at least one lower substituent.

[0307] In an embodiment, R 3C is hydrogen. In an embodiment, R 3C is unsubstituted C1-C4 alkyl. In an embodiment, R 3C is unsubstituted methyl. In an embodiment, R 3C is unsubstituted ethyl. In an embodiment, R 3C is unsubstituted propyl. In an embodiment, R 3C is unsubstituted n-propyl. In an embodiment, R 3C is unsubstituted isopropyl. In an embodiment, R 3C is unsubstituted butyl. In an embodiment, R 3C is unsubstituted n-butyl. In an embodiment, R 3Cis unsubstituted isobutyl. In an embodiment, R 3C is unsubstituted tert-butyl.

[0308] In an embodiment, R 4 is hydrogen. In an embodiment, R 4 is unsubstituted methyl.

[0309] In an embodiment, R 2 substituent and R 4 The substituted ring formed by the combination of substituents (for example, substituted heterocycloalkyl) is substituted with at least one substituent, a substituent with a restricted size or a lower substituent, and R 2 substituent and R 4 When the substituted ring formed by the combination of substituents is substituted with a plurality of groups selected from substituents, substituents with a restricted size and lower substituents, the substituents, substituents with a restricted size and / or lower substituents may each optionally be different. In an embodiment, R 2 substituent and R 4 When the substituted ring formed by the combination of substituents is substituted, it is substituted with at least one substituent. In an embodiment, R 2 substituent and R 4 When the substituted ring formed by the combination of substituents is substituted, it is substituted with at least one substituent with a restricted size. In an embodiment, R 2 substituent and R 4 When the substituted ring formed by the combination of substituents is substituted, it is substituted with at least one lower substituent.

[0310] In an embodiment, R 2 substituent and R 4 substituents combine to form a substituted or unsubstituted 3- to 8-membered heterocycloalkyl. In an embodiment, R 2 substituent and R 4 substituents combine to form a substituted or unsubstituted tetrahydrofuranyl.

[0311] In an embodiment, R 5 is -OH.

[0312] In an embodiment, substitution R 5A (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent, a size-restricted substituent, or a lower substituent, and substitution R 5A When is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents, the substituents, size-restricted substituents, and / or lower substituents may each optionally be different. In an embodiment, R 5A When is substituted, it is substituted with at least one substituent. In an embodiment, R 5A When is substituted, it is substituted with at least one size-restricted substituent. In an embodiment, R 5A When is substituted, it is substituted with at least one lower substituent.

[0313] In an embodiment, R 5A is hydrogen or substituted C1-C6 alkyl. In an embodiment, R 5A is hydrogen. In an embodiment, R 5A is unsubstituted C1-C4 alkyl. In an embodiment, R 5A is unsubstituted methyl. In an embodiment, R 5A is unsubstituted ethyl. In an embodiment, R 5A is unsubstituted propyl. In an embodiment, R 5A is unsubstituted n-propyl. In an embodiment, R 5A is unsubstituted isopropyl. In an embodiment, R 5A is unsubstituted butyl. In an embodiment, R 5A is unsubstituted n-butyl. In an embodiment, R 5A is unsubstituted isobutyl. In an embodiment, R 5A is unsubstituted tert-butyl. In an embodiment, R 5A is substituted C1-C6 alkyl. In an embodiment, R 5Ais dimethoxytrityl. In an embodiment, R 5A is

[0314]

Chemical formula

[0315] In an embodiment, substituted R 6 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, a size-restricted substituent or a lower substituent, and when substituted R 6 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents, the substituents, size-restricted substituents and / or lower substituents may each optionally be different. In an embodiment, when R 6 is substituted, it is substituted with at least one substituent. In an embodiment, when R 6 is substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when R 6 is substituted, it is substituted with at least one lower substituent. In an embodiment, R 6 is hydrogen. In an embodiment, R 6 is halogen. In an embodiment, R 6 is -F. In an embodiment, R 6 is -Cl. In an embodiment, R 6 is -Br. In an embodiment, R 6 is -I. In an embodiment, R 6 is -CCl3. In an embodiment, R 6 is -CBr3. In an embodiment, R 6 is -CF3. In an embodiment, R 6 is -CI3. In an embodiment, R 6 is -CH2Cl. In an embodiment, R 6 is -CH2Br. In an embodiment, R 6is -CH2F. In an embodiment, R 6 is -CH2I. In an embodiment, R 6 is -CHCl2. In an embodiment, R 6 is -CHBr2. In an embodiment, R 6 is -CHF2. In an embodiment, R 6 is -CHI2. In an embodiment, R 6 is -CN. In an embodiment, R 6 is -OH. In an embodiment, R 6 is -NH2. In an embodiment, R 6 is -COOH. In an embodiment, R 6 is -CONH2. In an embodiment, R 6 is -NO2. In an embodiment, R 6 is -SH. In an embodiment, R 6 is -SO3R A In an embodiment, R 6 is -SO3H. In an embodiment, R 6 is -SO2NH2. In an embodiment, R 6 is □NHNH2. In an embodiment, R 6 is □ONH2. In an embodiment, R 6 is □NHC(O)NH2. In an embodiment, R 6 is -NHSO2H. In an embodiment, R 6 is -NHC(O)H. In an embodiment, R 6 is -NHC(O)OH. In an embodiment, R 6 is -NHOH. In an embodiment, R 6 is -OCCl3. In an embodiment, R 6 is -OCBr3. In an embodiment, R 6 is -OCF3. In an embodiment, R 6 is -OCI3. In an embodiment, R 6 is -OCH2Cl. In an embodiment, R 6 is -OCH2Br. In an embodiment, R 6 is -OCH2F. In an embodiment, R 6 is -OCH2I. In an embodiment, R 6is -OCHCl2. In an embodiment, R 6 is -OCHBr2. In an embodiment, R 6 is -OCHF2. In an embodiment, R 6 is -OCHI2. In an embodiment, R 6 is -SF5. In an embodiment, R 6 is -N3. In an embodiment, R 6 is unsubstituted C1 - C4 alkyl. In an embodiment, R 6 is unsubstituted methyl. In an embodiment, R 6 is unsubstituted ethyl. In an embodiment, R 6 is unsubstituted propyl. In an embodiment, R 6 is unsubstituted n - propyl. In an embodiment, R 6 is unsubstituted isopropyl. In an embodiment, R 6 is unsubstituted butyl. In an embodiment, R 6 is unsubstituted n - butyl. In an embodiment, R 6 is unsubstituted isobutyl. In an embodiment, R 6 is unsubstituted tert - butyl. In an embodiment, R 6 is unsubstituted 2 - 6 - membered heteroalkyl. In an embodiment, R 6 is unsubstituted methoxy. In an embodiment, R 6 is unsubstituted ethoxy. In an embodiment, R 6 is unsubstituted propoxy. In an embodiment, R 6 is unsubstituted n - propoxy. In an embodiment, R 6 is unsubstituted isopropoxy. In an embodiment, R 6 is unsubstituted butoxy.

[0316] In an embodiment, substituted R 7 (for example, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, a size - restricted substituent or a lower substituent, and substituted R 7When it is substituted with a plurality of groups selected from a substituent, a substituent with a limited size, and a lower substituent, the substituent, the substituent with a limited size, and / or the lower substituent may each optionally be different. In an embodiment, R 7 when substituted, it is substituted with at least one substituent. In an embodiment, R 7 when substituted, it is substituted with at least one substituent with a limited size. In an embodiment, R 7 when substituted, it is substituted with at least one lower substituent.

[0317] In an embodiment, R 7 is hydrogen. In an embodiment, R 7 is a halogen. In an embodiment, R 7 is -F. In an embodiment, R 7 is -Cl. In an embodiment, R 7 is -Br. In an embodiment, R 7 is -I. In an embodiment, R 7 is -CCl3. In an embodiment, R 7 is -CBr3. In an embodiment, R 7 is -CF3. In an embodiment, R 7 is -CI3. In an embodiment, R 7 is -CH2Cl. In an embodiment, R 7 is -CH2Br. In an embodiment, R 7 is -CH2F. In an embodiment, R 7 is -CH2I. In an embodiment, R 7 is -CHCl2. In an embodiment, R 7 is -CHBr2. In an embodiment, R 7 is -CHF2. In an embodiment, R 7 is -CHI2. In an embodiment, R 7 is -CN. In an embodiment, R 7 is -OH. In an embodiment, R 7 is -NH2. In an embodiment, R 7 is -COOH. In an embodiment, R 7is -CONH2. In an embodiment, R 7 is -NO2. In an embodiment, R 7 is -SH. In an embodiment, R 7 is -SO3R A In an embodiment, R 7 is -SO3H. In an embodiment, R 7 is -SO2NH2. In an embodiment, R 7 is □NHNH2. In an embodiment, R 7 is □ONH2. In an embodiment, R 7 is □NHC(O)NH2. In an embodiment, R 7 is -NHSO2H. In an embodiment, R 7 is -NHC(O)H. In an embodiment, R 7 is -NHC(O)OH. In an embodiment, R 7 is -NHOH. In an embodiment, R 7 is -OCCl3. In an embodiment, R 7 is -OCBr3. In an embodiment, R 7 is -OCF3. In an embodiment, R 7 is -OCI3. In an embodiment, R 7 is -OCH2Cl. In an embodiment, R 7 is -OCH2Br. In an embodiment, R 7 is -OCH2F. In an embodiment, R 7 is -OCH2I. In an embodiment, R 7 is -OCHCl2. In an embodiment, R 7 is -OCHBr2. In an embodiment, R 7 is -OCHF2. In an embodiment, R 7 is -OCHI2. In an embodiment, R 7 is -SF5. In an embodiment, R 7 is -N3. In an embodiment, R 7 is unsubstituted C1 - C4 alkyl. In an embodiment, R 7 is unsubstituted methyl. In an embodiment, R 7 is unsubstituted ethyl. In an embodiment, R 7is unsubstituted propyl. In an embodiment, R 7 is unsubstituted n-propyl. In an embodiment, R 7 is unsubstituted isopropyl. In an embodiment, R 7 is unsubstituted butyl. In an embodiment, R 7 is unsubstituted n-butyl. In an embodiment, R 7 is unsubstituted isobutyl. In an embodiment, R 7 is unsubstituted tert-butyl. In an embodiment, R 7 is unsubstituted 2- to 6-membered heteroalkyl. In an embodiment, R 7 is unsubstituted methoxy. In an embodiment, R 7 is unsubstituted ethoxy. In an embodiment, R 7 is unsubstituted propoxy. In an embodiment, R 7 is unsubstituted n-propoxy. In an embodiment, R 7 is unsubstituted isopropoxy. In an embodiment, R 7 is unsubstituted butoxy.

[0318] In an embodiment, substituted R 8 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, a size-restricted substituent or a lower substituent, and when substituted R 8 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents, the substituents, size-restricted substituents and / or lower substituents may each optionally be different. In an embodiment, when R 8 is substituted, it is substituted with at least one substituent. In an embodiment, when R 8 is substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when R 8 is substituted, it is substituted with at least one lower substituent.

[0319] In an embodiment, R 8is hydrogen. In an embodiment, R 8 is a halogen. In an embodiment, R 8 is -F. In an embodiment, R 8 is -Cl. In an embodiment, R 8 is -Br. In an embodiment, R 8 is -I. In an embodiment, R 8 is -CCl3. In an embodiment, R 8 is -CBr3. In an embodiment, R 8 is -CF3. In an embodiment, R 8 is -CI3. In an embodiment, R 8 is -CH2Cl. In an embodiment, R 8 is -CH2Br. In an embodiment, R 8 is -CH2F. In an embodiment, R 8 is -CH2I. In an embodiment, R 8 is -CHCl2. In an embodiment, R 8 is -CHBr2. In an embodiment, R 8 is -CHF2. In an embodiment, R 8 is -CHI2. In an embodiment, R 8 is -CN. In an embodiment, R 8 is -OH. In an embodiment, R 8 is -NH2. In an embodiment, R 8 is -COOH. In an embodiment, R 8 is -CONH2. In an embodiment, R 8 is -NO2. In an embodiment, R 8 is -SH. In an embodiment, R 8 is -SO3R A . In an embodiment, R 8 is -SO3H. In an embodiment, R 8 is -SO2NH2. In an embodiment, R 8 is □NHNH2. In an embodiment, R 8 is □ONH2. In an embodiment, R 8 is □NHC(O)NH2. In an embodiment, R 8 is -NHSO2H. In an embodiment, R 8is -NHC(O)H. In an embodiment, R 8 is -NHC(O)OH. In an embodiment, R 8 is -NHOH. In an embodiment, R 8 is -OCCl3. In an embodiment, R 8 is -OCBr3. In an embodiment, R 8 is -OCF3. In an embodiment, R 8 is -OCI3. In an embodiment, R 8 is -OCH2Cl. In an embodiment, R 8 is -OCH2Br. In an embodiment, R 8 is -OCH2F. In an embodiment, R 8 is -OCH2I. In an embodiment, R 8 is -OCHCl2. In an embodiment, R 8 is -OCHBr2. In an embodiment, R 8 is -OCHF2. In an embodiment, R 8 is -OCHI2. In an embodiment, R 8 is -SF5. In an embodiment, R 8 is -N3. In an embodiment, R 8 is unsubstituted C1 - C4 alkyl. In an embodiment, R 8 is unsubstituted methyl. In an embodiment, R 8 is unsubstituted ethyl. In an embodiment, R 8 is unsubstituted propyl. In an embodiment, R 8 is unsubstituted n - propyl. In an embodiment, R 8 is unsubstituted isopropyl. In an embodiment, R 8 is unsubstituted butyl. In an embodiment, R 8 is unsubstituted n - butyl. In an embodiment, R 8 is unsubstituted isobutyl. In an embodiment, R 8 is unsubstituted tert - butyl. In an embodiment, R 8 is unsubstituted 2 - 6 - membered heteroalkyl. In an embodiment, R 8 is unsubstituted methoxy. In an embodiment, R 8 is unsubstituted ethoxy. In an embodiment, R8 is an unsubstituted propoxy. In an embodiment, R 8 is an unsubstituted n-propoxy. In an embodiment, R 8 is an unsubstituted isopropoxy. In an embodiment, R 8 is an unsubstituted butoxy.

[0320] In an embodiment, substituted R 9 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, a size-restricted substituent or a lower substituent, and when substituted R 9 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents, the substituents, size-restricted substituents and / or lower substituents may each optionally be different. In an embodiment, when R 9 is substituted, it is substituted with at least one substituent. In an embodiment, when R 9 is substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when R 9 is substituted, it is substituted with at least one lower substituent.

[0321] In an embodiment, R 9 is hydrogen. In an embodiment, R 9 is halogen. In an embodiment, R 9 is -F. In an embodiment, R 9 is -Cl. In an embodiment, R 9 is -Br. In an embodiment, R 9 is -I. In an embodiment, R 9 is -CCl3. In an embodiment, R 9 is -CBr3. In an embodiment, R 9 is -CF3. In an embodiment, R 9 is -CI3. In an embodiment, R 9 is -CH2Cl. In an embodiment, R 9is -CH2Br. In an embodiment, R 9 is -CH2F. In an embodiment, R 9 is -CH2I. In an embodiment, R 9 is -CHCl2. In an embodiment, R 9 is -CHBr2. In an embodiment, R 9 is -CHF2. In an embodiment, R 9 is -CHI2. In an embodiment, R 9 is -CN. In an embodiment, R 9 is -OH. In an embodiment, R 9 is -NH2. In an embodiment, R 9 is -COOH. In an embodiment, R 9 is -CONH2. In an embodiment, R 9 is -NO2. In an embodiment, R 9 is -SH. In an embodiment, R 9 is -SO3R A In an embodiment, R 9 is -SO3H. In an embodiment, R 9 is -SO2NH2. In an embodiment, R 9 is □NHNH2. In an embodiment, R 9 is □ONH2. In an embodiment, R 9 is □NHC(O)NH2. In an embodiment, R 9 is -NHSO2H. In an embodiment, R 9 is -NHC(O)H. In an embodiment, R 9 is -NHC(O)OH. In an embodiment, R 9 is -NHOH. In an embodiment, R 9 is -OCCl3. In an embodiment, R 9 is -OCBr3. In an embodiment, R 9 is -OCF3. In an embodiment, R 9 is -OCI3. In an embodiment, R 9 is -OCH2Cl. In an embodiment, R 9 is -OCH2Br. In an embodiment, R 9 is -OCH2F. In an embodiment, R 9is -OCH2I. In an embodiment, R 9 is -OCHCl2. In an embodiment, R 9 is -OCHBr2. In an embodiment, R 9 is -OCHF2. In an embodiment, R 9 is -OCHI2. In an embodiment, R 9 is -SF5. In an embodiment, R 9 is -N3. In an embodiment, R 9 is unsubstituted C1 - C4 alkyl. In an embodiment, R 9 is unsubstituted methyl. In an embodiment, R 9 is unsubstituted ethyl. In an embodiment, R 9 is unsubstituted propyl. In an embodiment, R 9 is unsubstituted n - propyl. In an embodiment, R 9 is unsubstituted isopropyl. In an embodiment, R 9 is unsubstituted butyl. In an embodiment, R 9 is unsubstituted n - butyl. In an embodiment, R 9 is unsubstituted isobutyl. In an embodiment, R 9 is unsubstituted tert - butyl. In an embodiment, R 9 is unsubstituted 2 - 6 - membered heteroalkyl. In an embodiment, R 9 is unsubstituted methoxy. In an embodiment, R 9 is unsubstituted ethoxy. In an embodiment, R 9 is unsubstituted propoxy. In an embodiment, R 9 is unsubstituted n - propoxy. In an embodiment, R 9 is unsubstituted isopropoxy. In an embodiment, R 9 is unsubstituted butoxy.

[0322] In an embodiment, substituted R A (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, a substituent with a limited size or a lower substituent, and substituted R AWhen it is substituted with a plurality of groups selected from substituents, substituents with restricted size, and lower substituents, the substituents, substituents with restricted size, and / or lower substituents may each optionally be different. In an embodiment, R A when substituted, is substituted with at least one substituent. In an embodiment, R A when substituted, is substituted with at least one substituent with restricted size. In an embodiment, R A when substituted, is substituted with at least one lower substituent.

[0323] In an embodiment, R A is hydrogen. In an embodiment, R A is unsubstituted C1-C4 alkyl. In an embodiment, R A is unsubstituted methyl. In an embodiment, R A is unsubstituted ethyl. In an embodiment, R A is unsubstituted propyl. In an embodiment, R A is unsubstituted n-propyl. In an embodiment, R A is unsubstituted isopropyl. In an embodiment, R A is unsubstituted butyl. In an embodiment, R A is unsubstituted n-butyl. In an embodiment, R A is unsubstituted isobutyl. In an embodiment, R A is unsubstituted tert-butyl.

[0324] In an embodiment, R 10 (e.g., substituted alkyl and / or substituted heteroalkyl) is substituted with at least one substituent, substituent with restricted size, or lower substituent, and when substituted R 10 is substituted with a plurality of groups selected from substituents, substituents with restricted size, and lower substituents, the substituents, substituents with restricted size, and / or lower substituents may each optionally be different. In an embodiment, R 10When it is replaced, it is substituted with at least one substituent. In an embodiment, R 10 When it is replaced, it is substituted with at least one substituent with a limited size. In an embodiment, R 10 When it is replaced, it is substituted with at least one lower substituent.

[0325] In an embodiment, R 10 is hydrogen. In an embodiment, R 10 is unsubstituted C1-C4 alkyl. In an embodiment, R 10 is unsubstituted methyl. In an embodiment, R 10 is unsubstituted ethyl. In an embodiment, R 10 is unsubstituted propyl. In an embodiment, R 10 is unsubstituted n-propyl. In an embodiment, R 10 is unsubstituted isopropyl. In an embodiment, R 10 is unsubstituted butyl. In an embodiment, R 10 is unsubstituted n-butyl. In an embodiment, R 10 is unsubstituted isobutyl. In an embodiment, R 10 is unsubstituted tert-butyl.

[0326] In an embodiment, the substituted ring (e.g., substituted heterocycloalkyl and / or substituted heteroaryl) formed by the R 1 substituent and the R 6 substituent is substituted with at least one substituent, a substituent with a limited size, or a lower substituent. When the substituted ring formed by the R 1 substituent and the R 6 substituent is substituted with a plurality of groups selected from substituents, substituents with a limited size, and lower substituents, the substituents, substituents with a limited size, and / or lower substituents may each optionally be different. In an embodiment, when the substituted ring formed by the R 1 substituent and the R 6 substituent is substituted, it is substituted with at least one substituent. In an embodiment, R1 The substituent and R 6 When the substituted ring formed by the bonding of the substituent is substituted, it is substituted with at least one substituent of limited size. In an embodiment, R 1 The substituent and R 6 When the substituted ring formed by the bonding of the substituent is substituted, it is substituted with at least one lower substituent.

[0327] In an embodiment, R 1 The substituent and R 6 The substituent bonds to form a substituted or unsubstituted 3- to 8-membered heterocycloalkyl. In an embodiment, R 1 The substituent and R 6 The substituent bonds to form a substituted or unsubstituted pyrrolidinyl. In an embodiment, R 1 The substituent and R 6 The substituent bonds to form an unsubstituted pyrrolidinyl. In an embodiment, R 1 The substituent and R 6 The substituent bonds to form a substituted or unsubstituted 5- to 6-membered heteroaryl.

[0328] In an embodiment, R 8 The substituent and R 10 The substituted ring formed by the bonding of the substituent (e.g., substituted heterocycloalkyl and / or substituted heteroaryl) is substituted with at least one substituent, substituent of limited size, or lower substituent, and R 8 The substituent and R 10 When the substituted ring formed by the bonding of the substituent is substituted with a plurality of groups selected from substituents, substituents of limited size, and lower substituents, the substituents, substituents of limited size, and / or lower substituents may each optionally be different. In an embodiment, R 8 The substituent and R 10 When the substituted ring formed by the bonding of the substituent is substituted, it is substituted with at least one substituent. In an embodiment, R 8 The substituent and R 10When the substituted ring formed by the attachment of a substituent is substituted, it is substituted with at least one substituent of limited size. In an embodiment, R 8 substituent and R 10 When the substituted ring formed by the attachment of a substituent is substituted, it is substituted with at least one lower substituent.

[0329] In an embodiment, R 8 substituent and R 10 substituents combine to form a substituted or unsubstituted 3- to 8-membered heterocycloalkyl. In an embodiment, R 8 substituent and R 10 substituents combine to form a substituted or unsubstituted pyrrolidinyl. In an embodiment, R 8 substituent and R 10 substituents combine to form an unsubstituted pyrrolidinyl. In an embodiment, R 8 substituent and R 10 substituents combine to form a substituted or unsubstituted 5- to 6-membered heteroaryl.

[0330] In an embodiment, the compound has the formula:

[0331]

Chem.

[0332] In an embodiment, the compound has the formula:

[0333]

Chem.

[0334] In an embodiment, the compound has the formula:

[0335]

Chem.

[0336] In an embodiment, the compound has the following formula:

[0337]

Chemical formula

[0338] In an embodiment, the compound has the following formula:

[0339]

Chemical formula

[0340] In an embodiment, the compound has the following formula:

[0341]

Chemical formula

[0342] In an embodiment, the compound has the following formula:

[0343]

Chemical formula

[0344] In an embodiment, the compound has the following formula:

[0345]

Chemical formula

[0346] In an embodiment, the compound has the following formula:

[0347]

Chemical formula

[0348] In an embodiment, the compound has the following formula:

[0349]

Chemical formula

[0350] In an embodiment, the compound has the following formula:

[0351]

Chemical formula

[0352] In an embodiment, the compound has the following formula:

[0353]

Chemical formula

[0354] In an embodiment, the compound has the following formula:

[0355]

Chemical formula

[0356] In an embodiment, the compound has the following formula:

[0357]

Chemical formula

[0358] In an embodiment, the compound has the following formula:

[0359]

Chemical formula

[0360] In an embodiment, the compound has the following formula:

[0361]

Chemical formula

[0362] In an embodiment, the compound has the following formula:

[0363]

Chemical formula

[0364] In an embodiment, the compound has the following formula:

[0365]

Chemical formula

[0366] In an embodiment, the compound has the following formula:

[0367]

Chemical formula

[0368] In an embodiment, the compound has the following formula:

[0369]

Chemical formula

[0370] In an embodiment, the compound has the following formula:

[0371]

Chemical formula

[0372] In an embodiment, the compound has the following formula:

[0373]

Chemical formula

[0374] In an embodiment, the compound has the following formula:

[0375]

Chemical formula

[0376] In an embodiment, the compound has the following formula:

[0377]

Chemical formula

[0378] In an embodiment, the compound has the following formula:

[0379]

Chemical formula

[0380] In an embodiment, the compound has the following formula:

[0381]

Chemical formula

[0382] In an embodiment, the compound has the following formula:

[0383]

Chemical formula

[0384] In an embodiment, the compound has the following formula:

[0385]

Chemical formula

[0386] In an embodiment, the compound has the following formula:

[0387]

Chemical formula

[0388] In an embodiment, the compound has the following formula:

[0389]

Chemical formula

[0390] In an embodiment, the compound has the following formula:

[0391]

Chemical formula

[0392] In an embodiment, the compound has the following formula:

[0393]

Chemical formula

[0394] In an embodiment, the compound is a compound described in this specification including the embodiment. In an embodiment, the compound is a compound described in this specification (e.g., in the Examples section, drawings, tables or claims).

[0395] Further details regarding labels / dyes Exemplary and non-limiting detectable labels that can be used with the embodiments described herein include, for example, fluorescein (e.g., 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 6-JOE, 6-carboxyfluorescein (6-FAM), VIC, FITC, 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxy-fluorescein (JOE)), 5-carboxy-1,4-dichloro-2’,7’-dichloro-fluorescein and 6-carboxy-1,4-dichloro-2’,7’-dichloro-fluorescein (TET), 5-carboxy-1,4-dichloro-2’,4’,5’,7’-tetra-chlorofluorescein and 6-carboxy-1,4-dichloro-2’,4’,5’,7’-tetra-chlorofluorescein, HEX, PET, NED, Oregon Green (e.g., 488, 500, 514)); pyrene (e.g., Cascade Blue; Alexa Fluor 405 coumarin (e.g., Pacific Blue, Atto 425, Alexa Fluor 350, Alexa Fluor 430); cyanine dyes (e.g., Cy dyes such as Cy3, Cy3.18, Cy3.5, Cy5, Cy5.18, Cy5.5, Cy7, etc.); rhodamine (e.g., 110, 123, B, B200, BB, BG, B extra, 5-carboxytetramethylrhodamine and 6-carboxytetramethylrhodamine (5-TAMRA, 6-TAMRA), 5-carboxyrhodamine 6G and 6-carboxyrhodamine 6G, Lissamine, Lissamine rhodamine B, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine) and 6-ROX (carboxy-X-rhodamine), sulfrorhodamine B can C, sulfrorhodamine G Extra, 5TAMRA and 6TAMRA (6-carboxytetramethyl-rhodamine), (TRITC), ABY, JUN, LIZ, RAD, RXJ, Texas Red; and Texas Red-X); Alexa Fluor fluorophores (a broad class that includes a number of dye types such as cyanines) (e.g., Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 500, Alexa 514, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa 610, Alexa 633, Alexa 635, Alexa 647, Alexa 660, Alexa 676, Alexa 680, Alexa 700, Alexa 750); FRET donor / acceptor pairs (e.g., fluorescein / fluorescein, fluorescein / rhodamine, fluorescein / cyanine, rhodamine / cyanine, fluorescein / Alexa Fluor, Alexa Fluor / rhodamine); and other types of dyes known to those skilled in the art.

[0396] The fluorophore label can be combined with quenchers such as dark fluorescent quencher (DFQ), black hole quencher (BHQ), Iowa Black, QSY7, QSY21 quencher, Dabsyl sulfonic acid / carboxylic acid quencher and Dabcel sulfonic acid / carboxylic acid quencher, and MGB-NFQ quencher. Examples of the fluorophore label can also include sulfonate derivatives of fluorescein dyes having SO3 instead of a carboxylate group, phosphoramidite forms of fluorescein, and / or phosphoramidite forms of Cy5.

[0397] Further details regarding amplification Amplification products obtained by using one or more embodiments described herein can be generated, detected, and / or analyzed on any suitable platform. In some embodiments, the nucleic acid target can be a single-stranded, double-stranded, or any other nucleic acid molecule of any size or conformation. The amplification processes described herein can include PCR (see, e.g., U.S. Patent No. 4,683,202). In some embodiments, the PCR is quantitative PCR (qPCR). In some embodiments, the PCR is endpoint PCR. In some embodiments, the PCR is digital PCR (dPCR).

[0398] In some embodiments, the amplification process includes reverse transcription PCR (RT-PCR). The disclosed methods can include, for example, subjecting the target nucleic acid to a reverse transcription reaction prior to amplification by PCR. In some embodiments, the amplification process includes one-step RT-PCR using one or more reverse transcriptases in combination with one or more DNA polymerases (e.g., in a single container or reaction volume).

[0399] Optionally, a particular qPCR assay sample can be placed in individual wells of a single array or multi-well plate, such as a TaqMan Array Card (e.g., Thermo Fisher Scientific, Waltham, Massachusetts; see catalog numbers 4346800 and 4342265) or a MicroAmp multi-well (e.g., 96-well, 384-well) reaction plate (e.g., Thermo Fisher Scientific, Waltham, Massachusetts; see catalog numbers 4346906, 4366932, 4306737, 4326659, and N8010560). Optionally, different qPCR assay samples present in different wells of the array or plate can be dried or lyophilized in situ prior to use, and the array or plate can be stored or transported. In some embodiments, the concepts described herein can be used for in situ hybridization applications that are not necessarily related to PCR.

[0400] Other amplification methods, such as loop-mediated isothermal amplification ( "LAMP (loop-mediated isothermal amplification)"), etc., and other isothermal methods are also contemplated for use with the assay embodiments described herein.

[0401] The components described herein that enable multiplexing using probes having detectable labels with spectral similarity can be provided in kit form together with one or more additional components to enable the amplification process. Such components include, for example, dNTPs, DNA polymerases, amplification buffers / reagents, master mix components known in the art, and other components known in the art to enable or assist nucleic acid amplification.

[0402] Implementation by computer system In some embodiments, at least a portion of the methods described herein can be implemented using one or more computer systems. In some examples, the techniques described herein can be represented by computer-executable instructions stored on one or more hardware storage devices. The computer-executable instructions can be executable by one or more processors for performing (or configuring the system to perform) the disclosed techniques. In some embodiments, the system can be configured to transmit the computer-executable instructions to a remote device to configure the remote device for performing the disclosed techniques.

[0403] In an exemplary embodiment, a computer system includes one or more processors and a memory storing one or more instructions that, when executed by the one or more processors, cause the one or more processors to perform the following process: obtaining emission (e.g., fluorescence) signal data related to a composite emission (e.g., fluorescence) signal from a first probe type including at least a first detectable label (e.g., fluorophore) and a second probe type including a second detectable label (e.g., fluorophore) having spectral similarity to and / or generating the same or substantially the same signal as the first detectable label (e.g., fluorophore), wherein the thermal and / or temporal characteristics of the first probe type and the second probe type are different, at multiple points in one or more cycles of an amplification process; and determining emission signal data related to an emission signal from a given probe type among at least the first probe type and the second probe type in one or more cycles of the amplification process, based at least in part on the emission signal data related to the composite emission signal of at least one of the first probe type and the second probe type and the thermal and / or temporal characteristics.

[0404] In some embodiments, using emission signal data related to a composite emission signal and first emission signal data as inputs for generating emission signal data related to an emission signal from a given probe type includes generating first emission signal data that has been transformed by applying a transformation (e.g., linear transformation) to the first emission signal data, and modifying the emission signal data related to the composite emission signal with the transformed first emission signal to generate emission signal data related to an emission signal from a given probe type.

[0405] In some embodiments, when one or more instructions are executed by one or more processors, the one or more processors are caused to quantify a first target associated with a first probe type based at least on first emission signal data, and to quantify a second target associated with a second probe type based at least on generated emission signal data associated with emission signals from a given probe type or types.

[0406] Some embodiments include one or more computer-readable media storing one or more instructions that, when executed by one or more processors of at least one computer device, cause the one or more processors to perform the above-described process or other computer-executable processes described herein.

[0407] A system for implementing the disclosed embodiments can include various components such as, by way of non-limiting example, processors (s), storage, sensors (s), I / O system (s), communication system (s), etc. The processor (s) can include one or more sets of electronic circuits including any number of logic units, registers, and / or control units to facilitate execution of computer-readable instructions (e.g., instructions forming a computer program). Such computer-readable instructions can be stored in storage. The storage can include physical system memory and can be volatile, non-volatile, or some combination thereof. Further, the storage can include local storage, remote storage (e.g., accessible via communication system (s), etc.), or some combination thereof.

[0408] Furthermore, the system may include or communicate with one or more I / O systems. The I / O system(s) may include any type of input or output device, such as, by way of non-limiting example, a display, touch screen, mouse, keyboard, controller, speaker, and / or others, but is not limited thereto. For example, the I / O system(s) may include a display system that may include any number of display panels, optics, laser scanning display assemblies, and / or other components.

[0409] The disclosed embodiments may also include physical media and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media can be any available media that can be accessed by a general-purpose or special-purpose computer system. Computer storage media (also known as "hardware storage devices") are RAM, ROM, EEPROM, CD-ROM, RAM-based solid state drives ("SSDs"), flash memory, phase-change memory ("PCM"), or other types of memory, or other optical disk storage, magnetic disk storage, or other magnetic storage devices, or any other media that can be used to store desired program code means in the form of computer-executable instructions, data, or data structures and that can be accessed by a general-purpose or special-purpose computer, i.e., a computer-readable hardware storage device.

[0410] Those skilled in the art will understand that the various embodiments can be implemented in a network computing environment with many types of computer system configurations, including personal computers, desktop computers, laptop computers, message processors, handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile phones, PDAs, pagers, routers, switches, wearable devices, and the like. The embodiments can also be implemented in a distributed system environment where a plurality of computer systems (e.g., local and remote computer systems) linked via a network (either by a wired data link, a wireless data link, or a combination of a wired data link and a wireless data link) execute tasks. In a distributed system environment, program modules can be in local memory storage devices and / or remote memory storage devices.

[0411] Alternatively or additionally, the functions described herein can be executed, at least in part, by one or more hardware logic components. For example, without limitation, exemplary types of hardware logic components that can be used include field-programmable gate arrays (FPGAs), program-specific integrated circuits (ASICs), application-specific standard products (ASSPs), system-on-a-chip systems (SOCs), complex programmable logic devices (CPLDs), central processing units (CPUs), graphics processing units (GPUs), and / or others.

[0412] As used herein, the terms "executable module", "executable component", "component", "module", or "engine" can refer to a hardware processing unit, or a software object, routine, or method that can be executed on one or more computer systems. The different components, modules, engines, and services described herein may be implemented as objects or processors (e.g., as separate threads) executing on one or more computer systems.

Example

[0413] FIG. 6A shows the fluorescence signal versus the number of cycles measured at the annealing / extension temperature (65° C. in this example) and denaturation temperature (95° C. in this example) using TaqMan probe compositions and EF probe compositions.

[0414] FIGS. 6B-6D show the results of a qPCR duplex assay test measuring the fluorescence signal in the FAM detection channel, in which the TaqMan probe and the EF probe were designed to generate fluorescence signals with spectral similarity (e.g., and detectable in the same detection channel) (FIG. 6B), or without spectral similarity (e.g., and detectable in different detection channels) (FIGS. 6C and 6D). In the assay shown in FIG. 6B, both the TaqMan probe and the EF probe were labeled with FAM. In the assay shown in FIG. 6C, the TaqMan probe was labeled with ABY and the EF probe was labeled with FAM. In the assay shown in FIG. 6D, the TaqMan probe was labeled with FAM and the EF probe was labeled with ABY. The composition of the reaction mixture, template DNA concentration, and amplification conditions were kept the same among the assays in other respects.

[0415] In FIG. 6B, the top row shows the fluorescence signal of the FAM channel versus the number of cycles measured at the denaturation temperature (95° C. in this example). This signal is expected to mainly contain fluorescence generated by the TaqMan probe label (cleaved from the probe). The second row from the top shows the fluorescence signal versus the number of cycles measured at the denaturation temperature (95° C. in this example) and corrected by a linear function that correlates the measured value of the TaqMan probe at 95° C. with the measured value at 65° C. This signal is expected to contain fluorescence generated by the TaqMan probe label but not significant fluorescence from the EF probe label. The third row shows the fluorescence signal versus the number of cycles measured at the annealing / extension temperature (65° C. in this example). This signal is expected to contain fluorescence generated by both the TaqMan probe label (cleaved from the probe) and the EF probe label (incorporated into the double-stranded amplicon). The bottom row shows the resolved fluorescence signal determined by subtracting the second row signal from the third row signal. This signal is expected to estimate the fluorescence generated by the EF probe label separately from the fluorescence due to the TaqMan probe label.

[0416] In FIG. 6C, the top row, second row, third row, and bottom row represent the same signal measurement types as in FIG. 6B using a TaqMan probe labeled with ABY and an EF probe labeled with FAM. As shown in the top row and the second row, the EF probe label (FAM) generated slight (essentially baseline-level) fluorescence in the FAM channel at the denaturation temperature. In this assay, since the TaqMan probe and the EF probe were labeled with different labels, the bottom row shows a resolved signal of the EF probe label that essentially matches the signal of the EF probe at the annealing / extension temperature in the FAM channel (third row).

[0417] In FIG. 6D, the topmost, second, third, and bottommost layers are of the same type as in FIG. 6B, but represent the signal measurement type using a TaqMan probe labeled with FAM and an EF probe labeled with ABY. Most of the fluorescence signals in the first layer are generated by TaqMan probe labeling (those cleaved from the probe). The second layer shows the fluorescence signal versus the cycle number, measured at the denaturation temperature (95° C. in this example) and corrected by a linear function that correlates the measured value of the TaqMan probe at 95° C. with the measured value at 65° C. In this assay, since the TaqMan probe and the EF probe are labeled with different labels, the TaqMan signal (second layer) derived from the FAM channel essentially coincides with the signal measured at the annealing / extension temperature in the FAM channel (third layer), and the signal after decomposition of the EF probe labeling in the FAM channel (bottommost layer) is essentially 0.

[0418] FIGS. 6B to 6D show the results obtained from a qPCR assay, but the same principle shown can be extended to an endpoint PCR assay that shows similar results. Specifically, a correction factor is applied to the signals measured under a series of denaturation conditions (e.g., denaturation temperature), and the derived signals are subtracted from the signals measured under a series of extension conditions (e.g., extension temperature) to obtain the signal derived from the second probe (non-cleavable), and the measured signals from the series of denaturation conditions represent the signals from the first probe (cleavable probe).

[0419] FIG. 6E compares the decomposed EF-related fluorescence signal (ΔRn) after baseline adjustment (bottommost layer of FIG. 6B) with the EF-related fluorescence signal (ΔRn) after baseline adjustment that represents the direct measurement of the fluorescence of the EF probe labeling in the FAM channel (bottommost layer of FIG. 6C). The results showed a close correlation between the decomposed signal and the measured signal. Therefore, the results showed that the fluorescence signals caused by different probe types within the same detection channel can be decomposed separately.

[0420] Figure 6F compares the derived TaqMan-related fluorescence signal (ΔRn) after baseline adjustment (second row of Figure 6B) with the derived TaqMan-related fluorescence signal (ΔRn) after baseline adjustment (second row of Figure 6D) in the FAM channel. From the results, it was shown that there is a close correlation between TaqMan signals derived from separate assays that label the EF probe similarly (Figure 6B) or label the EF probe with different dyes (Figure 6D).

[0421] Figure 7 shows the results of an assay test that includes five different detection channels / dyes, where four detection channels use the corresponding TaqMan probe and EF probe (each channel has a different dye common to the TaqMan probe and EF probe within that channel), and one channel uses only the TaqMan probe (AF647 is noted in the index of Figure 7). From the results, it can be seen that the fluorescence signals of different probe types can be determined independently, and that a 9-plex reaction can be effectively carried out using five detection channels.

[0422] Figure 8 is a plot comparing the endpoint signals of the reaction volume at 65°C and 95°C after the dPCR process. As shown, the signals are classified into distinguishable clusters. The clusters can be estimated using cluster analysis algorithms known in the art. In Figure 8, the "EF" cluster represents the fraction where signals are obtained at the annealing / extension temperature but few signals are obtained at the denaturation temperature, the "T" cluster represents the fraction where signals are obtained at both the annealing / extension temperature and the denaturation temperature, and the "T+EF" cluster represents the fraction where signals are obtained at the denaturation temperature and an increase in signal is seen at the annealing / extension temperature. The total number of fractions in which the TaqMan probe generated a signal is equal to the number of clusters T plus the number of clusters T+EF, and the total number of fractions in which the EF probe generated a signal is equal to the number of clusters EF plus the number of clusters T+EF. Then, using standard dPCR techniques, the concentrations of the first target and the second target in the sample can be estimated.

[0423] The cluster analysis technique of FIG. 8 is one embodiment of a technique for analyzing signals obtained in a dPCR process. Regarding other data analysis techniques that can be used to decompose signal data obtained in a multiplex dPCR process that utilizes a detectable label having spectral similarity, in accordance with the present disclosure.

[0424] The present disclosure includes embodiments represented by the following items, without being limited thereto. Item 1: A method for detecting nucleic acid in a sample, comprising: (i) A step of preparing a reaction mixture, wherein the reaction mixture comprises: at least a part of the sample, a first probe detectably labeled with a first label configured to generate a first luminescence signal, a second probe detectably labeled with a second label configured to generate a second luminescence signal, and the first probe and the second probe have different sequences, the first label and the second label are the same and / or generate substantially the same luminescence, (ii) A step of subjecting the reaction mixture to an amplification process including a first set of reaction conditions and a second set of reaction conditions, wherein the first set of reaction conditions is different from the second set of reaction conditions; (iii) Measuring the luminescence signal in the first set of reaction conditions to determine the presence of a first nucleic acid target and / or a second nucleic acid target in the sample, wherein the luminescence signal in the first set of reaction conditions correlates with a specific interaction or lack of interaction between the first probe and the first nucleic acid target, Measuring the luminescence signal in the second set of reaction conditions, wherein the luminescence signal in the second set of reaction conditions correlates with a specific interaction or lack of interaction between the first probe and the first nucleic acid target, and a specific interaction or lack of interaction between the second probe and the second nucleic acid target, and ​ estimating the presence and / or amount of each of a first nucleic acid target and a second nucleic acid target, determined by, a method comprising.

[0425] Item 2: A method for detecting nucleic acid in a sample, (i) preparing a reaction mixture, the reaction mixture comprising at least a portion of the sample, a first probe detectably labeled with a first label configured to generate a first luminescence signal indicative of the presence or absence of a first nucleic acid target, a second probe detectably labeled with a second label configured to generate a second luminescence signal indicative of the presence or absence of a second nucleic acid target, and the first probe and the second probe having different sequences, the first label and the second label being the same and / or generating substantially the same luminescence signal, (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions, (iii) determining the presence or absence and / or amount of each of the first nucleic acid target and / or the second nucleic acid target in the reaction mixture by measuring a first total luminescence signal comprising any first luminescence signal, if present, and any second luminescence signal, if present, in the first set of reaction conditions, measuring a second total luminescence signal comprising any first luminescence signal, if present, and any second luminescence signal, if present, in the second set of reaction conditions, and estimating the first luminescence signal and / or the second luminescence signal based on the first total luminescence signal and the second total luminescence signal, determined by, a method comprising.

[0426] Item 3: The method according to Item 1 or 2, wherein the first luminescence signal and the second luminescence signal are the first fluorescence signal and the second fluorescence signal, both the first probe and the second probe are excited at the same wavelength, and / or both the first probe and the second probe are excited during detection of their respective first fluorescence signal and second fluorescence signal.

[0427] Item 4: The luminescence signal is a fluorescence signal, the step of measuring the fluorescence signal under the first set of reaction conditions includes measuring a total signal including the first fluorescence signal and the second fluorescence signal under the first set of reaction conditions to obtain a first total fluorescence signal, the step of measuring the fluorescence signal under the second set of reaction conditions includes measuring a total signal including the first fluorescence signal and the second fluorescence signal under the second set of reaction conditions to obtain a second total fluorescence signal, and the step of estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target includes estimating the first fluorescence signal and / or the second fluorescence signal based on the first total fluorescence signal and the second total fluorescence signal. the method according to any one of Items 1 to 3.

[0428] Item 5: The method according to Item 4, wherein the second fluorescence signal differs more significantly between the first set of reaction conditions and the second set of reaction conditions than the first fluorescence signal differs between the first set of reaction conditions and the second set of reaction conditions.

[0429] Item 6: The first total fluorescence value includes (i) fluorescence from the first label that is released as a result of cleavage of the first label after hybridization of the first probe to the first amplicon, which is free in the reaction mixture and not quenched, and (ii) background fluorescence of the second label. The second total fluorescence value is based on the fluorescence from the second label, where the fluorescence from the second label exceeds (i) the fluorescence from the first label that is released as a result of cleavage of the first label after hybridization of the first probe to the first amplicon and that is free and not quenched in the reaction mixture, and (ii) the background fluorescence of the second label that is released as a result of hybridization of the second probe to the second amplicon. The method according to any one of items 4 to 5.

[0430] Item 7: Calculating the amount of the first nucleic acid target based on the first fluorescence signal; Calculating the amount of the second nucleic acid target based on the second fluorescence signal; The method according to any one of items 4 to 6, further comprising:

[0431] Item 8: That the first fluorescence signal exceeds the background level in both the first set of reaction conditions and the second set of reaction conditions indicates the presence of the first nucleic acid target in the reaction mixture. The method according to any one of items 4 to 7.

[0432] Item 9: That the second fluorescence signal exceeds the background level in the second set of reaction conditions but not in the first set of reaction conditions indicates the presence of the second nucleic acid target in the reaction mixture. The method according to any one of items 4 to 8.

[0433] Item 10: The first set of reaction conditions includes the first measurement temperature at which the first fluorescence signal is measured, the second set of reaction conditions includes the second measurement temperature at which the second fluorescence signal is measured, and the second measurement temperature is different from the first measurement temperature. The method according to any one of items 5 to 8.

[0434] Item 11: The method according to item 10, wherein the first measurement temperature and the second measurement temperature differ by at least about 10 °C, about 15 °C, about 20 °C, about 25 °C or about 30 °C or more.

[0435] Claim 12: The method according to claim 10 or 11, wherein at least one of the first measured temperature or the second measured temperature is a denaturation temperature at which DNA in the reaction mixture denatures, such as in the range of about 80 °C or higher.

[0436] Claim 13: The method according to any one of claims 1 to 12, wherein the reaction mixture is subjected to a plurality of amplification cycles in an amplification process, and each amplification cycle includes a first set of reaction conditions and a second set of reaction conditions.

[0437] Claim 14: The method according to any one of claims 1 to 13, wherein the amplification process includes thermal cycling.

[0438] Claim 15: The method according to claim 14, wherein the step of subjecting the reaction mixture to the first set of reaction conditions includes performing thermal cycling on the reaction mixture at a first temperature sufficient to cause denaturation of the first amplicon and the second amplicon.

[0439] Claim 16: The method according to claim 15, wherein the step of subjecting the reaction mixture to the second set of reaction conditions includes performing thermal cycling on the reaction mixture at a second temperature sufficient to cause annealing and / or extension of the first nucleic acid target and the second nucleic acid target to form the first amplicon and the second amplicon, respectively, and the second temperature is lower than the first temperature.

[0440] Claim 17: The method according to any one of claims 1 to 16, wherein the first probe is a cleavable probe.

[0441] Claim 18: The method according to claim 17, wherein the first emission signal increases as the cleavable probe is cleaved during the annealing / extension step.

[0442] Item 19: The method according to item 17 or 18, wherein the first probe comprises a fluorophore and a quencher, and the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved in the annealing / extension step of the amplification process.

[0443] Item 20: The method according to item 19, wherein the first probe is a TaqMan probe.

[0444] Item 21: The method according to any one of items 1 to 20, wherein the second probe is a non-cleavable probe.

[0445] Item 22: The method according to item 21, wherein the second probe comprises a stem-loop portion configured to form a stem-loop structure when the second probe is single-stranded.

[0446] Item 23: The method according to item 21 or 22, wherein the second probe comprises a fluorophore and a quencher that are arranged apart from each other such that the fluorophore is quenched when the second probe is single-stranded and the fluorophore is not quenched when the second probe is incorporated into a double-stranded amplicon.

[0447] Item 24: The method according to item 23, wherein the fluorophore is located at or near the 5'-end of the second probe and the quencher is on the 3'-side of the fluorophore.

[0448] Item 25: The method according to item 23 or 24, wherein both the fluorophore and the quencher are arranged in or near the stem-loop portion of the second probe.

[0449] Item 26: The method according to any one of Items 1 to 25, further comprising: a first primer pair complementary to a first nucleic acid target of a nucleic acid or its complement, wherein the first nucleic acid target is configured to generate a first amplicon to which a first probe can hybridize; and a second primer pair complementary to a second nucleic acid target of a nucleic acid or its complement, wherein the second nucleic acid target is configured to generate a second amplicon to which a second probe can hybridize.

[0450] Item 27: The method according to Item 26, wherein the second primer pair includes a primer having a tail.

[0451] Item 28: The method according to Item 27, wherein the tail forms the 5' end of the primer having the tail.

[0452] Item 29: The method according to Item 27 or 28, wherein the second probe can hybridize to a tail or its complement.

[0453] Item 30: The method according to any one of Items 20 to 29, wherein the amplification process uses a series of thermal cycling steps including at least three different target temperatures.

[0454] Item 31: The method according to Item 30, wherein the amplification process includes a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

[0455] Item 32: The method according to Item 31, wherein a first series of denaturation steps and annealing / extension steps are performed at a first annealing / extension temperature, and a second series of denaturation steps and annealing / extension steps are performed at a second annealing / extension temperature different from the first annealing / extension temperature.

[0456] Item 33: The method according to Item 32, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

[0457] Claim 34: The method according to claim 32 or 33, wherein the first series of denaturation steps and annealing / elongation steps are cycled more times than the second series of denaturation steps and annealing / elongation steps.

[0458] Claim 35: The method according to any one of claims 32 to 34, wherein the amplification process further comprises a third series of denaturation steps and annealing / elongation steps performed at a third annealing / elongation temperature.

[0459] Claim 36: The method according to claim 35, wherein the third annealing / elongation temperature is the same as the first annealing / elongation temperature.

[0460] Claim 37: The method according to claim 35 or 36, wherein the third series of denaturation steps and annealing / elongation steps are cycled more times than the first series of denaturation steps and annealing / elongation steps.

[0461] Claim 38: The method according to any one of claims 32 to 37, wherein the denaturation temperature is the same in each series of denaturation steps.

[0462] Claim 39: The method according to any one of claims 27 to 38, wherein the second primer pair further comprises a primer without a tail, and the concentration of the primer with a tail in the reaction mixture is different from the concentration of the primer without a tail in the reaction mixture.

[0463] Claim 40: The method according to claim 39, wherein the concentration of the primer without a tail is higher than the concentration of the primer with a tail.

[0464] Claim 41: The method according to claim 40, wherein the concentration of the primer without a tail is about 2× to about 30×, or about 5× to about 25×, or about 10× to about 20× the concentration of the primer with a tail.

[0465] Item 42: The method according to any one of Items 39 to 41, wherein a second probe is prepared at a concentration different from the concentration of the primer having a tail and the concentration of the primer without a tail.

[0466] Item 43: The method according to Item 42, wherein the second probe is prepared at a concentration higher than the concentration of the primer having a tail.

[0467] Item 44: The method according to Item 42 or 43, wherein the second probe is prepared at a concentration lower than the concentration of the primer without a tail.

[0468] Item 45: The method according to any one of Items 42 to 44, wherein the second probe is prepared at a concentration of about 2× to about 10× the concentration of the primer having a tail, or about 3× to about 7.5× the concentration of the primer having a tail.

[0469] Item 46: The melting temperature (T m ) of the first probe and the T m of the second probe are within about 8 °C, or within about 6 °C, or within about 4 °C, or within about 2 °C of each other. The method according to any one of Items 1 to 45.

[0470] Item 47: The method according to any one of Items 1 to 48, wherein the amplification process cycles between at least two target temperatures over a plurality of cycles of the amplification process.

[0471] Item 48: The method according to Item 47, wherein the amplification process cycles between at least two target temperatures over at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cycles of the amplification process.

[0472] Item 49: The method according to any one of Items 1 to 48, further comprising a step of distributing the reaction mixture into a plurality of reaction volumes, and wherein the amplification process is a digital PCR (dPCR) process.

[0473] Item 50: The step of measuring the luminescence signal under a first set of reaction conditions includes performing a first endpoint measurement by measuring the luminescence signal when or almost when the step of subjecting the reaction mixture to the first set of reaction conditions is completed. The step of measuring the luminescence signal under a second set of reaction conditions includes performing a second endpoint measurement by measuring the luminescence signal when or almost when the step of subjecting the reaction mixture to the second set of reaction conditions is completed. The method according to Item 49.

[0474] Item 51: The step of estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target includes a step of classifying a plurality of reaction volumes according to the luminescence signal measured in the first endpoint measurement and according to the luminescence signal measured in the second endpoint measurement, and a step of determining the number of reaction volumes in which the first probe showed activity and the number of reaction volumes in which the second probe showed activity based on the classification. The method according to Item 50.

[0475] Item 52: The step of measuring the luminescence signal under a first set of reaction conditions includes a step of measuring the luminescence signal in the denaturation stage of the endpoint cycle of the amplification process. The step of measuring the luminescence signal under a second set of reaction conditions includes a step of measuring the luminescence signal in the annealing and / or extension stage of the endpoint cycle of the amplification process. The method according to any one of Items 1 to 48.

[0476] Item 53: The method according to any one of Items 1 to 48 or Item 52, wherein the amplification process is an endpoint PCR process.

[0477] Item 54: A method for detecting nucleic acid in a sample, comprising: A step of preparing a reaction mixture, wherein the reaction mixture comprises: A primer pair complementary to a nucleic acid target for generating an amplicon or its complement, and A non-cleavable probe configured to hybridize to the amplicon and containing a detectable label configured to generate a luminescence signal corresponding to the amount of the generated amplicon, A step of subjecting the reaction mixture to an amplification process to generate an amplicon, wherein the label generates luminescence even if the non-cleavable probe is not cleaved in the amplification process, and the amplification process uses a series of thermal cycle steps including at least three different target temperatures, A step of measuring the luminescence signal from the non-cleavable probe, and A method comprising the above steps.

[0478] Item 55: The method according to Item 54, further comprising a step of quantifying the amount of the nucleic acid target based on the measured luminescence signal.

[0479] Item 56: The method according to Item 54 or 55, wherein the non-cleavable probe comprises a stem-loop portion capable of forming a stem-loop structure when the non-cleavable probe is single-stranded.

[0480] Item 57: The method according to any one of Items 54 to 56, wherein the non-cleavable probe comprises a fluorophore and a quencher that are arranged separately such that the fluorophore is quenched when the non-cleavable probe is single-stranded, but becomes luminescent when the probe is incorporated into a double-stranded amplicon.

[0481] Item 58: The method according to Item 57, wherein the fluorophore is located at or near the 5'-end of the probe, and the quencher is on the 3'-side of the fluorophore.

[0482] Item 59: The method according to Item 57 or 58, wherein both the fluorophore and the quencher are in or near the stem-loop portion of the probe.

[0483] Item 60: The method according to any one of Items 54 to 59, wherein the primer pair includes a primer having a tail.

[0484] Item 61: The method according to Item 60, wherein the tail forms the 5'-end of the primer having the tail.

[0485] Item 62: The method according to Item 60 or 61, wherein the non-cleavable probe is configured to hybridize to the tail or its complement.

[0486] Item 63: The method according to Item 62, wherein the 3'-portion of the non-cleavable probe is configured to hybridize to the tail or its complement.

[0487] Item 64: The method according to any one of Items 54 to 63, wherein the amplification process includes a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

[0488] Item 65: The method according to Item 64, wherein the first series of denaturation steps and annealing / extension steps are performed at a first annealing / extension temperature, and the second series of denaturation steps and annealing / extension steps are performed at a second annealing / extension temperature different from the first annealing / extension temperature.

[0489] Item 66: The method according to Item 65, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

[0490] Item 67: The method according to Item 65 or 66, wherein the first series of denaturation steps and annealing / extension steps are cycled more times than the second series of denaturation steps and annealing / extension steps.

[0491] Claim 68: The method according to any one of claims 65 to 67, wherein the amplification process further comprises a third series of denaturation steps and annealing / elongation steps carried out using a third annealing / elongation temperature.

[0492] Claim 69: The method according to claim 68, wherein the third annealing / elongation temperature is the same as the first annealing / elongation temperature.

[0493] Claim 70: The method according to claim 68 or 69, wherein the third series of denaturation steps and annealing / elongation steps are cycled more times than the first series of denaturation steps and annealing / elongation steps.

[0494] Claim 71: The method according to any one of claims 65 to 70, wherein the denaturation temperature is the same in each series of denaturation steps.

[0495] Claim 72: The method according to any one of claims 65 to 71, wherein the primer pair further comprises a primer without a tail, and the concentration of the primer with a tail in the reaction mixture is different from the concentration of the primer without a tail.

[0496] Claim 73: The method according to claim 72, wherein the primer without a tail is prepared at a higher concentration than the primer with a tail.

[0497] Claim 74: The method according to claim 73, wherein the primer without a tail is prepared at a concentration of about 2× to about 30× the concentration of the primer with a tail, or about 5× to about 25× the concentration of the primer with a tail, or about 10× to about 20× the concentration of the primer with a tail.

[0498] Claim 75: The method according to any one of claims 72 to 74, wherein the concentration of the non-cleavable probe in the reaction mixture is different from the concentration of the primer with a tail and the concentration of the primer without a tail in the reaction mixture.

[0499] Item 76: The method according to item 75, wherein the concentration of the non-cleavable probe in the reaction mixture is higher than the concentration of the primer having a tail in the reaction mixture.

[0500] Item 77: The method according to item 75 or 76, wherein the non-cleavable probe is prepared at a concentration lower than the concentration of the primer without a tail.

[0501] Item 78: The method according to any one of items 75 to 77, wherein the non-cleavable probe is prepared at a concentration of about 2× to about 10× the concentration of the primer without a tail, or at a concentration of about 3× to about 7.5× the concentration of the primer without a tail.

[0502] Item 79: The reaction mixture comprises A primer pair complementary to a second nucleic acid target or its complement for generating a second amplicon, and A cleavable probe configured to hybridize to the second amplicon and comprising a detectable label configured to generate a luminescence signal corresponding to the amount of the generated second amplicon, And further comprises By subjecting the reaction mixture to an amplification process, a second amplicon is generated, and in the amplification process, cleavage of the cleavable probe generates luminescence of the detectable label of the cleavable probe, The method further comprises the step of measuring the luminescence signal from the cleavable probe, The method according to any one of items 54 or 55.

[0503] Item 80: The method according to item 81, further comprising the step of quantifying the amount of the second nucleic acid target based on the measured luminescence signal.

[0504] Item 81: A method for detecting nucleic acid in a sample, comprising A step of preparing a reaction mixture, wherein the reaction mixture comprises A primer pair targeting a nucleic acid target for generating an amplicon, the primer pair including a primed primer and a non-tailed primer prepared at different concentrations, and A non-cleavable probe that is detectably labeled and configured to hybridize to the amplicon to generate a fluorescent signal corresponding to the amount of the generated amplicon, and Including steps, A step of subjecting a reaction mixture to an amplification process to generate an amplicon, wherein the non-cleavable probe generates luminescence even if it is not cleaved in the amplification process, A step of measuring the luminescence signal from the non-cleavable probe, and Including methods.

[0505] Item 82: The method according to item 81, wherein the non-tailed primer is prepared at a higher concentration than the primed primer.

[0506] Item 83: The method according to item 82, wherein the non-tailed primer is prepared at a concentration of about 2× to about 30× the concentration of the primed primer, or about 5× to about 25× the concentration of the primed primer, or about 10× to about 20× the concentration of the primed primer.

[0507] Item 84: The method according to any one of items 80 to 83, wherein the non-cleavable probe is prepared at a concentration different from the concentration of the primed primer and the concentration of the non-tailed primer.

[0508] Item 85: The method according to item 84, wherein the non-cleavable probe is prepared at a concentration higher than the concentration of the primed primer.

[0509] Item 86: The method according to item 84 or 84, wherein the non-cleavable probe is prepared at a concentration lower than the concentration of the non-tailed primer.

[0510] Item 87: The method according to any one of Items 84 to 86, wherein a non-cleavable probe is prepared at a concentration of about 2× to about 10× the concentration of a primer without a tail, or at a concentration of about 3× to about 7.5× the concentration of a primer without a tail.

[0511] Item 88: A reaction mixture A primer pair for targeting a second nucleic acid target different from the nucleic acid target and generating a second amplicon, the primer pair including a primer with a tail and a primer without a tail prepared at different concentrations, A detectable and cleavable probe configured to hybridize to the second amplicon and generate a luminescence signal corresponding to the amount of the generated second amplicon, and further including By subjecting the reaction mixture to an amplification process, a second amplicon is generated, and the cleavable probe generates a luminescence signal without being cleaved in the amplification process, The method further includes the step of measuring the luminescence signal from the cleavable probe, The method according to Item 81.

[0512] Item 89: A method for detecting the presence or amount of a first target and / or a second target in a reaction mixture, including the step of including a first probe and a second probe in the reaction mixture, the first probe including a first label capable of specifically interacting with the first target and generating a first detectable signal, and the second probe including a second label capable of specifically interacting with the second target and generating a second detectable signal, the step of specifically interacting the first probe and the second probe with any first target and second target, respectively, in the reaction mixture, Measuring a first total signal through an optical filter under a first set of conditions, the first total signal including a first detectable signal and a second detectable signal from a first label and a second label, wherein under the first set of conditions, the first detectable signal increases as a result of specific interaction between a first probe and a first target, while the second detectable signal does not increase as a result of specific interaction between a second probe and a second target; Measuring a second total signal through the same optical filter under a second set of conditions, the second total signal including a first detectable signal and a second detectable signal from a first label and a second label, wherein under the second set of conditions, the second detectable signal increases as a result of specific interaction between a second probe and a second target; Evaluating the presence and / or amount of the first target and / or the second target by estimating the first detectable signal and the second detectable signal based on both the first total signal and the second total signal; A method comprising the above steps.

[0513] Item 90: The method according to Item 89, wherein the first label and the second label are the same and / or generate substantially the same fluorescence.

[0514] Item 91: The method according to Item 89 or Item 90, wherein the second fluorescence signal differs more significantly between the first set of reaction conditions and the second set of conditions than the first fluorescence signal does between the first set of reaction conditions and the second set of conditions.

[0515] Item 92: The method according to any one of Items 89 to 91, wherein the first probe is a cleavable probe.

[0516] Item 93: The method according to Item 92, wherein the increase in the first detectable signal indicates that the cleavable probe has been cleaved.

[0517] Item 94: The method according to item 92 or 93, wherein the first probe comprises a fluorophore and a quencher, and the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved.

[0518] Item 95: The method according to item 94, wherein the first probe is a TaqMan probe.

[0519] Item 96: The method according to any one of items 89 to 95, wherein the second probe is a non-cleavable probe.

[0520] Item 97: The method according to item 96, wherein the second probe comprises a stem-loop portion capable of forming a stem-loop structure when the second probe is single-stranded.

[0521] Item 98: The method according to item 96 or 97, wherein the second label of the second probe is a fluorophore, and the second probe further comprises a quencher, wherein the fluorophore is quenched when the second probe is single-stranded, but the fluorophore becomes capable of emitting light when the second probe is incorporated into a double-stranded nucleic acid and is arranged apart therefrom.

[0522] Item 99: The method according to item 98, wherein the fluorophore is located at or near the 5'-end of the second probe, and the quencher is on the 3'-side of the fluorophore.

[0523] Item 100: The method according to item 98 or 99, wherein both the fluorophore and the quencher are arranged at or near the stem-loop portion of the second probe.

[0524] Item 101: The melting temperature (T m ) of the first probe and the T m of the second probe are within about 8 °C, or about 6 °C, or about 4 °C, or about 2 °C of each other. The method according to any one of items 89 to 100.

[0525] Item 102: The method according to any one of Items 89 to 101, wherein the first set of conditions includes a first measurement temperature at which a first fluorescence signal is measured, and the second set of conditions includes a second different measurement temperature at which a second fluorescence signal is measured.

[0526] Item 103: The method according to Item 102, wherein the first measurement temperature and the second measurement temperature differ by at least about 10°C, about 15°C, about 20°C, about 25°C, or about 30°C or more.

[0527] Item 104: The method according to Item 102 or 103, wherein at least one of the first measurement temperature or the second measurement temperature is a denaturation temperature at which DNA in the reaction mixture denatures, such as about 90°C or more.

[0528] Item 105: The method according to any one of Items 89 to 104, further including thermal cycling of the reaction mixture between two target temperatures over a plurality of cycles.

[0529] Item 106: The method according to Item 105, wherein the thermal cycling cycles between two target temperatures over at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cycle.

[0530] Item 107: The method according to any one of Items 54 to 106, wherein the step of measuring the signal is performed in an endpoint thermal cycle of the amplification process.

[0531] Item 108: The method according to any one of Items 1, 2 or 86, wherein the first probe is configured to generate a cumulative signal over different stages of a cycle of an amplification process, and the second probe is configured to generate a transient signal at different stages of a cycle of an amplification process.

[0532] Item 109: The second probe is a compound having the following formula:

[0533]

Chemical formula

[0534]

Chemical formula

[0535] Claim 110: When the second probe is single-stranded, L 5 forms a stem-loop structure, the method according to claim 109.

[0536] Claim 111: R 50 is a fluorophore, and Q and R 50 are arranged apart from each other such that when the second probe is single-stranded, R 50 is quenched, and when the second probe is incorporated into the double-stranded amplicon, it is not quenched, the method according to claim 109.

[0537] Claim 112: The method according to claim 111, wherein both Q and R 50 are arranged in or near the stem-loop portion of the second probe.

[0538] Claim 113: The method according to claim 109, wherein L 5 contains 11 to 30 nucleotides.

[0539] Item 114: L 5 The method according to item 109, wherein it contains 19 to 23 nucleotides.

[0540] Item 115: L 5 The method according to item 109, wherein it contains 4 to 14 nucleotides.

[0541] Item 116: L 5 The method according to item 109, wherein it contains 6 to 12 nucleotides.

[0542] Item 117: The method according to item 109, wherein the nucleotide is a DNA nucleotide.

[0543] Item 118: The method according to item 109, wherein the nucleotide is an RNA molecule.

[0544] Item 119: The method according to item 109, wherein the compound has the following formula:

[0545]

Chemical formula

[0546] Item 120: The method according to item 109, wherein the compound has the following formula:

[0547]

Chemical formula

[0548] Item 121: The method according to item 109, wherein the compound has the following formula:

[0549]

Chemical formula

[0550] Item 122: The method according to item 109, wherein the compound has the following formula:

[0551] [Chemical formula] The method according to claim 109, having

[0552] Claim 123: The method according to claim 109, wherein B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent xanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or divalent 5-hydroxymethylcytosine or a derivative thereof.

[0553] Claim 124: The method according to claim 109, wherein B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, or divalent uracil or a derivative thereof.

[0554] Claim 125: The compound has the following formula:

[0555] [Chemical formula] The method according to claim 109, having

[0556] Claim 126: The compound has the following formula:

[0557] [Chemical formula] The method according to claim 109, having

[0558] Claim 127: The compound has the following formula:

[0559] [Chemical formula] The method according to claim 109, having

[0560] Claim 128: The compound has the following formula:

[0561]

Chemical formula

[0562] Claim 129: L 1 is L 101 -L 102 -L 103 -L 104 -L 105 and L 101 L 102 L 103 L 104 and L 105 are independently a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. The method according to claim 109.

[0563] Claim 130: L 101 is -S(O)2-. The method according to claim 129.

[0564] Claim 131: L 102 is unsubstituted 3- to 8-membered heterocycloalkyl. The method according to claim 129.

[0565] Claim 132: L 102 is unsubstituted piperidinyl. The method according to claim 129.

[0566] Claim 133: L 102 is

[0567] [Chemical formula] The method according to claim 129, wherein it is .

[0568] Claim 134: L 103 The method according to claim 129, wherein it is -C(O)NH-.

[0569] Claim 135: L 104 The method according to claim 129, wherein it is unsubstituted C1-C10 alkylene, unsubstituted 2-6 membered heteroalkylene or unsubstituted phenylene.

[0570] Claim 136: L 104 wherein it is unsubstituted n-hexylene,

[0571] [Chemical formula] The method according to claim 129, wherein it is .

[0572] Claim 137: L 105 wherein it is unsubstituted C1-C 10 alkylene, substituted or unsubstituted 2-8 membered heteroalkylene, or unsubstituted 5-10 membered heteroarylene, the method according to claim 129.

[0573] Claim 138: L 105 wherein it is

[0574] [Chemical formula] The method according to claim 129, wherein it is .

[0575] Claim 139: L 1 wherein it is

[0576] [Chemical formula] The method according to claim 129

[0577] Claim 140: L 50 The method according to claim 109, wherein is a substituted 2- to 10-membered heteroalkylene

[0578] Claim 141: L 50 is

[0579]

Chemical formula

[0580] Claim 142: R 50 The method according to claim 109, wherein is a fluorescent moiety

[0581] Claim 143: R 50 The method according to claim 142, wherein is the monovalent form of FAM, the monovalent form of VIC, the monovalent form of ABY, the monovalent form of JUN, the monovalent form of AF647, the monovalent form of Cy5, the monovalent form of AF676 or the monovalent form of Cy5.5

[0582] Claim 144: R 2 The method according to claim 109, wherein is hydrogen or -OH

[0583] Claim 145: The method according to claim 109, wherein R2 is hydrogen

[0584] Claim 146: R 30 The method according to any one of claims 109 to 145, wherein is -OH

[0585] Claim 147: R 30 is

[0586]

Chemical formula

[0587] Item 148: The 3'-blocking moiety is monovalent dideoxycytidine (3'ddC), monovalent dideoxyadenosine (ddA), 3'-reverse dT, 3'-amino modifier, monovalent QSY7, monovalent QSY21, monovalent QSY9, monovalent BHQ1, monovalent BHQ2, monovalent BHQ3, monovalent Dabcyl, monovalent Dabsyl, monovalent Eclipse, monovalent BBQ-650, monovalent Iowa Black RQ, monovalent Iowa Black FQ,

[0588]

Chemical formula

[0589] Item 149: A composition for detecting nucleic acid in a sample, A first probe detectably labeled with a first label configured to generate a first luminescence signal, A second probe detectably labeled with a second label configured to generate a second luminescence signal, comprising wherein the first probe and the second probe have different sequences, the first label and the second label are the same and / or generate substantially the same luminescence, under a first set of conditions, the first label generates a first luminescence signal that increases as a result of the specific interaction between the first probe and the first nucleic acid target, and the second label generates a second luminescence signal that does not increase as a result of the specific interaction between the second probe and the second nucleic acid target, under a second set of conditions different from the first set of conditions, the second luminescence signal increases as a result of the specific interaction between the second probe and the second nucleic acid target, the composition.

[0590] Item 150: The composition according to item 149, wherein the first probe is a cleavable probe.

[0591] Item 151: The composition according to any one of items 149 and 150, wherein the first probe comprises a fluorophore and a quencher, and the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved in the annealing / extension step of the amplification process.

[0592] Item 152: The composition according to any one of items 149 to 151, wherein the first probe is a TaqMan probe.

[0593] Item 153: The composition according to any one of items 149 to 152, wherein the second probe is a non-cleavable probe.

[0594] Item 154: The composition according to any one of items 149 to 152, wherein the second probe comprises a stem-loop portion configured to form a stem-loop structure when the second probe is single-stranded.

[0595] Item 155: The composition according to any one of items 153 or 154, wherein the second probe comprises a fluorophore and a quencher that are arranged apart from each other such that the fluorophore is quenched when the second probe is single-stranded and the fluorophore is not quenched when the second probe is incorporated into the double-stranded amplicon.

[0596] Item 156: The composition according to item 155, wherein the fluorophore is located at or near the 5'-end of the second probe and the quencher is on the 3'-side of the fluorophore.

[0597] Item 157: The composition according to any one of items 155 or 156, wherein both the fluorophore and the quencher are arranged at or near the stem-loop portion of the second probe.

[0598] Item 158: The composition according to any one of Items 149 to 157, further comprising: a first primer pair complementary to a first nucleic acid target of a nucleic acid or its complement, wherein the first nucleic acid target is configured to generate a first amplicon to which a first probe can hybridize; and a second primer pair complementary to a second nucleic acid target of a nucleic acid or its complement, wherein the second nucleic acid target is configured to generate a second amplicon to which a second probe can hybridize.

[0599] Item 159: The composition according to Item 158, wherein the second primer pair comprises a primer having a tail.

[0600] Item 160: The composition according to Item 159, wherein the tail forms the 5' end of the primer having the tail.

[0601] Item 161: The composition according to any one of Items 159 or 160, wherein the second probe can hybridize to a tail or its complement.

[0602] Item 162: The composition according to any one of Items 159 to 161, wherein the second primer pair further comprises a primer without a tail, and the concentration of the primer having a tail in the reaction mixture is different from the concentration of the primer without a tail in the reaction mixture.

[0603] Item 163: The composition according to Item 162, wherein the concentration of the primer without a tail is higher than the concentration of the primer having a tail.

[0604] Item 164: The composition according to Item 163, wherein the concentration of the primer without a tail is about 2× to about 30×, or about 5× to about 25×, or about 10× to about 20× the concentration of the primer having a tail.

[0605] Item 165: The composition according to any one of Items 162 to 164, wherein the second probe is prepared at a concentration different from the concentration of the primer having a tail and the concentration of the primer without a tail.

[0606] Item 166: The composition according to Item 165, wherein the second probe is prepared at a concentration higher than the concentration of the primer having a tail.

[0607] Item 167: The composition according to any one of Items 165 or 166, wherein the second probe is prepared at a concentration lower than the concentration of the primer without a tail.

[0608] Item 168: The composition according to any one of Items 165 to 167, wherein the second probe is prepared at a concentration of about 2× to about 10× the concentration of the primer having a tail, or about 3× to about 7.5× the concentration of the primer having a tail.

[0609] Item 169: A primer pair complementary to a nucleic acid target for generating an amplicon or its complement, An uncleavable probe configured to hybridize to the amplicon, the uncleavable probe comprising a detectable label configured to produce a luminescence signal corresponding to the amount of the generated amplicon, comprising In an amplification process, the detectable label includes a series of thermal cycling steps including at least two different target temperatures, and luminescence is generated even if the uncleavable probe is not cleaved. Composition.

[0610] Item 170: The composition according to Item 169, wherein the composition is a reaction mixture.

[0611] Item 171: The composition according to Item 169 or 170, comprising a stem-loop portion capable of forming a stem-loop structure when the uncleavable probe is single-stranded.

[0612] Item 172: The composition according to any one of Items 159 to 170, comprising a fluorophore and a quencher, which are arranged separately such that when the non-cleavable probe is single-stranded, the fluorophore is quenched, but when the probe is incorporated into a double-stranded amplicon, it becomes capable of emitting light.

[0613] Item 173: The composition according to Item 172, wherein the fluorophore is located at or near the 5'-end of the probe, and the quencher is on the 3'-side of the fluorophore.

[0614] Item 174: The composition according to Item 172, wherein both the fluorophore and the quencher are at or near the stem-loop portion of the probe.

[0615] Item 175: The composition according to any one of Items 169 to 174, wherein the primer pair includes a primer having a tail.

[0616] Item 176: The composition according to Item 175, wherein the tail forms the 5'-end of the primer having the tail.

[0617] Item 177: The composition according to Item 175 or 176, wherein the non-cleavable probe is configured to hybridize to the tail or its complement.

[0618] Item 178: The composition according to Item 177, wherein the 3'-portion of the non-cleavable probe is configured to hybridize to the tail or its complement.

[0619] Item 179: The composition according to any one of Items 169, wherein the primer pair includes a primer having a tail prepared at a different concentration and a primer without a tail.

[0620] Item 180: The composition according to Item 179, wherein the primer without a tail is prepared at a higher concentration than the primer having a tail.

[0621] Item 181: The composition according to item 180, wherein the primer without a tail is prepared at a concentration of about 2× to about 30× the concentration of the primer with a tail, or about 5× to about 25× the concentration of the primer with a tail, or about 10× to about 20× the concentration of the primer with a tail.

[0622] Item 182: The composition according to item 179, wherein the concentration of the non-cleavable probe in the reaction mixture is higher than the concentration of the primer with a tail in the reaction mixture.

[0623] Item 183: The composition according to item 179, wherein the non-cleavable probe is prepared at a concentration lower than the concentration of the primer without a tail.

[0624] Item 184: The method according to any one of items 183, wherein the non-cleavable probe is prepared at a concentration of about 2× to about 10× the concentration of the primer without a tail, or about 3× to about 7.5× the concentration of the primer without a tail.

[0625] Item 185: The reaction mixture comprises a primer pair complementary to a second nucleic acid target or its complement for generating a second amplicon, and a cleavable probe configured to hybridize to the second amplicon and comprising a detectable label configured to produce a luminescence signal corresponding to the amount of the generated second amplicon, and further comprises By subjecting the reaction mixture to an amplification process, a second amplicon is generated, and in the amplification process, cleavage of the cleavable probe causes the detectable label of the cleavable probe to generate luminescence, The method further comprises the step of measuring the luminescence signal from the cleavable probe, The composition according to any one of items 169 to 184.

[0626] Item 186: A kit comprising the composition according to any one of Items 149 to 185.

[0627] Those skilled in the art will understand that various other changes can be made to the structure, arrangement, method, materials, etc. without departing from the scope and principle of operation of the present disclosure. By way of example, in various embodiments, methods for performing the detection of two different targets using a first probe and a second probe having spectral similarity are described, but it is also possible to use other numbers of targets and probes in those methods to increase the multiplexity of the entire amplification and detection assay.

[0628] Those skilled in the art will understand how to combine any feature or operation disclosed herein with any one or a combination of any other features and operations disclosed herein. Further, the content or feature of any one of the drawings may be combined with or used in relation to any content or feature used in any of the other drawings. In this regard, the content disclosed in any one of the drawings is not mutually exclusive of the content of any of the other drawings, but rather can be combined.

[0629] The described embodiments should be considered in all respects to be illustrative and not restrictive. All changes that come within the meaning and range equivalent to the claims should be embraced within their scope.

Claims

**Claim 1** A method for detecting nucleic acid in a sample, comprising: (i) preparing a reaction mixture, wherein the reaction mixture comprises at least a part of the sample, a first probe detectably labeled with a first label configured to generate a first luminescence signal, a second probe detectably labeled with a second label configured to generate a second luminescence signal, wherein the first probe and the second probe have different sequences, the first label and the second label are the same and / or generate substantially the same luminescence; (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, wherein the first set of reaction conditions is different from the second set of reaction conditions; (iii) measuring the luminescence signal under the first set of reaction conditions, wherein the luminescence signal under the first set of reaction conditions correlates with the specific interaction or lack of interaction between the first probe and the first nucleic acid target; measuring the luminescence signal under the second set of reaction conditions, wherein the luminescence signal under the second set of reaction conditions correlates with the specific interaction or lack of interaction between the first probe and the first nucleic acid target and the specific interaction or lack of interaction between the second probe and the second nucleic acid target; and estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target, thereby determining the method. A method comprising the above steps. **Claim 2** A method for detecting nucleic acid in a sample, comprising: (i) preparing a reaction mixture, wherein the reaction mixture comprises at least a part of the sample, a first probe detectably labeled with a first label configured to generate a first luminescence signal indicating the presence or absence of a first nucleic acid target, a second probe detectably labeled with a second label configured to generate a second luminescence signal indicating the presence or absence of a second nucleic acid target, wherein the first probe and the second probe have different sequences, the first label and the second label are the same and / or generate substantially the same luminescence signal; ​ Step of supplying the reaction mixture to an amplification process, which includes a first set of reaction conditions and a second set of reaction conditions different from the first set of reaction conditions; Step of determining the presence, absence and / or amount of each of the first nucleic acid target and / or the second nucleic acid target in the reaction mixture; Step of measuring a first total luminescence signal, which includes any first luminescence signal if present and any second luminescence signal if present, in the first set of reaction conditions; Step of measuring a second total luminescence signal, which includes any first luminescence signal if present and any second luminescence signal if present, in the second set of reaction conditions; and Step of estimating the first luminescence signal and / or the second luminescence signal based on the first total luminescence signal and the second total luminescence signal; Step of determining by; Method including the above steps.

3. The method according to claim 1 or 2, wherein the first luminescence signal and the second luminescence signal are a first fluorescence signal and a second fluorescence signal, both the first probe and the second probe are excited at the same wavelength, and / or both the first probe and the second probe are excited during detection of the respective first fluorescence signal and second fluorescence signal.

4. The luminescence signal is a fluorescence signal. The step of measuring the fluorescence signal in the first set of reaction conditions includes measuring a total signal including the first fluorescence signal and the second fluorescence signal in the first set of reaction conditions to obtain a first total fluorescence signal. The step of measuring the fluorescence signal in the second set of reaction conditions includes measuring a total signal including the first fluorescence signal and the second fluorescence signal in the second set of reaction conditions to obtain a second total fluorescence signal. And the step of estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target includes the step of estimating the first fluorescence signal and / or the second fluorescence signal based on the first total fluorescence signal and the second total fluorescence signal. The method according to any one of claims 1 to 3.

5. ​ ​ ​ ​ The method according to claim 4, wherein the second fluorescence signal differs more significantly between the first set of reaction conditions and the second set of reaction conditions than the degree to which the first fluorescence signal differs between the first set of reaction conditions and the second set of reaction conditions.

6. The first total fluorescence value includes (i) fluorescence from the first label that is free in the reaction mixture and not quenched and is emitted as a result of cleavage of the first label after hybridization of the first probe to the first amplicon, and (ii) background fluorescence of the second label. The second total fluorescence value is based on fluorescence from the second label and is greater than (i) fluorescence from the first label that is free in the reaction mixture and not quenched and is emitted as a result of cleavage of the first label after hybridization of the first probe to the first amplicon, and (ii) background fluorescence of the second label that is emitted as a result of hybridization of the second probe to the second amplicon. The method according to any one of claims 4 or 5.

7. A step of calculating the amount of the first nucleic acid target based on the first fluorescence signal; A step of calculating the amount of the second nucleic acid target based on the second fluorescence signal; The method according to any one of claims 4 to 6, further comprising.

8. The method according to any one of claims 4 to 7, wherein the fact that the first fluorescence signal exceeds the background level under both the first set of reaction conditions and the second set of reaction conditions indicates the presence of the first nucleic acid target in the reaction mixture.

9. The method according to any one of claims 4 to 8, wherein the fact that the second fluorescence signal exceeds the background level under the second set of reaction conditions rather than the first set of reaction conditions indicates the presence of the second nucleic acid target in the reaction mixture.

10. The method according to any one of claims 5 to 8, wherein the first set of reaction conditions includes a first measurement temperature at which the first fluorescence signal is measured, the second set of reaction conditions includes a second measurement temperature at which the second fluorescence signal is measured, and the second measurement temperature is different from the first measurement temperature.

11. The method according to claim 10, wherein the first measured temperature and the second measured temperature differ by at least about 10 °C or more, about 15 °C or more, about 20 °C or more, about 25 °C or more, or about 30 °C or more.

12. The method according to claim 10 or claim 11, wherein at least one of the first measured temperature or the second measured temperature is a denaturation temperature at which DNA in the reaction mixture denatures, such as in the range of about 80 °C or higher.

13. The method according to any one of claims 1 to 12, wherein the reaction mixture is subjected to a plurality of amplification cycles in the amplification process, and each of the amplification cycles includes the first set of reaction conditions and the second set of reaction conditions.

14. The method according to any one of claims 1 to 13, wherein the amplification process includes thermal cycling.

15. The method according to claim 14, wherein the step of subjecting the reaction mixture to the first set of reaction conditions includes performing thermal cycling on the reaction mixture at a first temperature sufficient to cause denaturation of the first amplicon and the second amplicon.

16. The method according to claim 15, wherein the step of subjecting the reaction mixture to the second set of reaction conditions includes performing thermal cycling on the reaction mixture at a second temperature sufficient to cause annealing and / or extension of the first nucleic acid target and the second nucleic acid target to form the first amplicon and the second amplicon, respectively, and the second temperature is lower than the first temperature.

17. The method according to any one of claims 1 to 16, wherein the first probe is a cleavable probe.

18. The method according to claim 17, wherein the first emission signal increases as the cleavable probe is cleaved during the annealing / extension stage.

19. The method according to any one of claims 17 or 18, wherein the first probe includes a fluorophore and a quencher, and the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved during the annealing / extension stage of the amplification process.

20. The method according to claim 19, wherein the first probe is a TaqMan probe.

21. The method according to any one of claims 1 to 20, wherein the second probe is a non-cleavable probe.

22. The method according to claim 21, wherein the second probe includes a stem-loop portion configured to form a stem-loop structure when the second probe is single-stranded.

23. The method according to claim 21 or claim 22, wherein the second probe comprises a fluorophore and a quencher, and the fluorophore is quenched when the second probe is single-stranded and the fluorophore is not quenched when the second probe is incorporated into a double-stranded amplicon, and the fluorophore and the quencher are arranged apart from each other.

24. The method according to claim 23, wherein the fluorophore is located at or near the 5' end of the second probe, and the quencher is on the 3' side of the fluorophore.

25. The method according to claim 23 or claim 24, wherein both the fluorophore and the quencher are arranged in or near the stem-loop portion of the second probe.

26. The method according to any one of claims 1 to 25, wherein the reaction mixture further comprises a first primer pair complementary to a first nucleic acid target of the nucleic acid or its complement, wherein the first nucleic acid target is configured to generate a first amplicon to which the first probe can hybridize, and a second primer pair complementary to a second nucleic acid target of the nucleic acid or its complement, wherein the second nucleic acid target is configured to generate a second amplicon to which the second probe can hybridize.

27. The method according to claim 26, wherein the second primer pair includes a primer having a tail.

28. The method according to claim 27, wherein the tail forms the 5' end of the primer having the tail.

29. The method according to claim 27 or claim 28, wherein the second probe can hybridize to the tail or its complement.

30. The method according to any one of claims 20 to 29, wherein the amplification process uses a series of thermal cycling steps including at least three different target temperatures.

31. The method according to claim 30, wherein the amplification process includes a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

32. The method according to claim 31, wherein the first series of denaturation steps and annealing / elongation steps are carried out at a first annealing / elongation temperature, and the second series of denaturation steps and annealing / elongation steps are carried out at a second annealing / elongation temperature different from the first annealing / elongation temperature.

33. The method according to claim 32, wherein the first annealing / elongation temperature is higher than the second annealing / elongation temperature.

34. The method according to claim 32 or claim 33, wherein the first series of denaturation steps and annealing / elongation steps are cycled more times than the second series of denaturation steps and annealing / elongation steps.

35. The method according to any one of claims 32 to 34, wherein the amplification process further includes a third series of denaturation steps and annealing / elongation steps carried out using a third annealing / elongation temperature.

36. The method according to claim 35, wherein the third annealing / elongation temperature is the same as the first annealing / elongation temperature.

37. The method according to claim 35 or claim 36, wherein the third series of denaturation steps and annealing / elongation steps are cycled more times than the first series of denaturation steps and annealing / elongation steps.

38. The method according to any one of claims 32 to 37, wherein the denaturation temperature is the same in each series of denaturation steps.

39. The method according to any one of claims 27 to 38, wherein the second primer pair further includes a primer without a tail, and the concentration of the primer with a tail in the reaction mixture is different from the concentration of the primer without a tail in the reaction mixture.

40. The method according to claim 39, wherein the concentration of the primer without a tail is higher than the concentration of the primer with a tail.

41. The method according to claim 40, wherein the concentration of the primer without a tail is about 2× to about 30×, or about 5× to about 25×, or about 10× to about 20× the concentration of the primer with a tail.

42. The method according to any one of claims 39 to 41, wherein the second probe is prepared at a concentration different from the concentration of the primer with a tail and the concentration of the primer without a tail.

43. The method according to claim 42, wherein the second probe is prepared at a concentration higher than the concentration of the primer having the tail.

44. The method according to claim 42 or 43, wherein the second probe is prepared at a concentration lower than the concentration of the primer without the tail.

45. The method according to any one of claims 42 to 44, wherein the second probe is prepared at a concentration of about 2× to about 10× the concentration of the primer having the tail, or about 3× to about 7.5× the concentration of the primer having the tail.

46. The melting temperature (T ｍ ) of the first probe and the T ｍ of the second probe are within about 8 °C, or within about 6 °C, or within about 4 °C, or within about 2 °C of each other. The method according to any one of claims 1 to 45.

47. The method according to any one of claims 1 to 48, wherein the amplification process cycles between at least two target temperatures over a plurality of cycles of the amplification process.

48. The method according to claim 47, wherein the amplification process cycles between at least two target temperatures over at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cycles of the amplification process.

49. The method according to any one of claims 1 to 48, further comprising the step of dispensing the reaction mixture into a plurality of reaction volumes, and wherein the amplification process is a digital PCR (dPCR) process.

50. The step of measuring the luminescence signal under the first set of reaction conditions includes measuring the luminescence signal at the time when or substantially at the time when the step of subjecting the reaction mixture to the first set of reaction conditions is completed, to perform a first endpoint measurement, and the step of measuring the luminescence signal under the second set of reaction conditions includes measuring the luminescence signal at the time when or substantially at the time when the step of subjecting the reaction mixture to the second set of reaction conditions is completed, to perform a second endpoint measurement. The method according to claim 49.

51. The step of estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target comprises classifying the plurality of reaction volumes according to the emission signal measured in the first endpoint measurement and according to the emission signal measured in the second endpoint measurement; determining, based on the classification, the number of the plurality of reaction volumes in which the first probe was active and the number of the plurality of reaction volumes in which the second probe was active; The method according to claim 50, comprising:

52. The method according to any one of claims 1 to 48, wherein the step of measuring the emission signal under the first set of reaction conditions includes measuring the emission signal in the denaturation stage of the endpoint cycle of the amplification process, and the step of measuring the emission signal under the second set of reaction conditions includes measuring the emission signal in the annealing and / or extension stage of the endpoint cycle of the amplification process.

53. The method according to any one of claims 1 to 48 or claim 52, wherein the amplification process is an endpoint PCR process.

54. A method for detecting nucleic acid in a sample, comprising: preparing a reaction mixture, the reaction mixture comprising: a primer pair complementary to a nucleic acid target for generating an amplicon or its complement; a non-cleavable probe configured to hybridize to the amplicon and configured to generate an emission signal corresponding to the amount of the generated amplicon, the non-cleavable probe comprising a detectable label; including; subjecting the reaction mixture to an amplification process to generate the amplicon, wherein the label generates light even if the non-cleavable probe is not cleaved in the amplification process, and the amplification process uses a series of thermal cycle steps including at least three different target temperatures; measuring the emission signal from the non-cleavable probe; including.

55. The method according to claim 54, further comprising quantifying the amount of the nucleic acid target based on the measured emission signal.

56. The method according to claim 54 or claim 55, wherein the non-cleavable probe can form a stem-loop structure when the non-cleavable probe is single-stranded, the non-cleavable probe comprising a stem-loop portion.

57. The non-cleavable probe is a fluorophore and a quencher, which are arranged separately such that when the non-cleavable probe is single-stranded, the fluorophore is quenched, but when the probe is incorporated into a double-stranded amplicon, it can emit light. The method according to any one of claims 54 to 56, comprising a fluorophore and a quencher.

58. The method according to claim 57, wherein the fluorophore is located at or near the 5' end of the probe, and the quencher is on the 3' side of the fluorophore.

59. The method according to claim 57 or claim 58, wherein both the fluorophore and the quencher are in or near the stem-loop portion of the probe.

60. The method according to any one of claims 54 to 59, wherein the primer pair includes a primer having a tail.

61. The method according to claim 60, wherein the tail forms the 5' end of the primer having the tail.

62. The method according to claim 60 or claim 61, wherein the non-cleavable probe is configured to hybridize to the tail or its complement.

63. The method according to claim 62, wherein the 3' portion of the non-cleavable probe is configured to hybridize to the tail or its complement.

64. The method according to any one of claims 54 to 63, wherein the amplification process includes a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

65. The method according to claim 64, wherein a first series of denaturation steps and annealing / extension steps are performed at a first annealing / extension temperature, and a second series of denaturation steps and annealing / extension steps are performed at a second annealing / extension temperature different from the first annealing / extension temperature.

66. The method according to claim 65, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

67. The method according to claim 65 or claim 66, wherein the first series of denaturation steps and annealing / extension steps are cycled more times than the second series of denaturation steps and annealing / extension steps.

68. The method according to any one of claims 65 to 67, wherein the amplification process further comprises a third series of denaturation steps and annealing / extension steps carried out using a third annealing / extension temperature.

69. The method according to claim 68, wherein the third annealing / extension temperature is the same as the first annealing / extension temperature.

70. The method according to claim 68 or claim 69, wherein the third series of denaturation steps and annealing / extension steps are cycled more times than the first series of denaturation steps and annealing / extension steps.

71. The method according to any one of claims 65 to 70, wherein the denaturation temperature is the same in each series of denaturation steps.

72. The method according to any one of claims 65 to 71, wherein the primer pair further comprises a primer without a tail, and the concentration of the primer with a tail in the reaction mixture is different from the concentration of the primer without a tail in the reaction mixture.

73. The method according to claim 72, wherein the primer without a tail is prepared at a higher concentration than the primer with a tail.

74. The method according to claim 73, wherein the primer without a tail is prepared at a concentration of about 2× to about 30×, or about 5× to about 25×, or about 10× to about 20× the concentration of the primer with a tail.

75. The method according to any one of claims 72 to 74, wherein the concentration of the non-cleavable probe in the reaction mixture is different from the concentration of the primer with a tail and the concentration of the primer without a tail in the reaction mixture.

76. The method according to claim 75, wherein the concentration of the non-cleavable probe in the reaction mixture is higher than the concentration of the primer with a tail in the reaction mixture.

77. The method according to claim 75 or claim 76, wherein the non-cleavable probe is prepared at a concentration lower than the concentration of the primer without a tail.

78. The method according to any one of claims 75 to 77, wherein the non-cleavable probe is prepared at a concentration of about 2× to about 10×, or about 3× to about 7.5× the concentration of the primer without a tail.

79. The reaction mixture is A primer pair complementary to a second nucleic acid target or its complement for generating a second amplicon, A cleavable probe configured to hybridize to the second amplicon and comprising a detectable label configured to produce a luminescence signal corresponding to the amount of the generated second amplicon, the cleavable probe, further comprising, By subjecting the reaction mixture to the amplification process, a second amplicon is generated, and in the amplification process, cleavage of the cleavable probe causes the detectable label of the cleavable probe to generate luminescence, the method further comprising the step of measuring the luminescence signal from the cleavable probe, The method according to any one of claims 54 or 55.

80. The method according to claim 81, further comprising the step of quantifying the amount of the second nucleic acid target based on the measured luminescence signal.

81. A method for detecting nucleic acids in a sample, comprising: A step of preparing a reaction mixture, wherein the reaction mixture A primer pair that targets a nucleic acid target for generating an amplicon and includes a primer with a tail and a primer without a tail, prepared at different concentrations, A non-cleavable probe that is detectably labeled and configured to hybridize to the amplicon and generate a fluorescence signal corresponding to the amount of the generated amplicon, including, A step of subjecting the reaction mixture to an amplification process to generate the amplicon, wherein the non-cleavable probe generates luminescence even if it is not cleaved in the amplification process, measuring the luminescence signal from the non-cleavable probe; including,

82. The method according to claim 81, wherein the primer without a tail is prepared at a higher concentration than the primer with a tail.

83. The method according to claim 82, wherein the primer without a tail is prepared at a concentration of about 2× to about 30× the concentration of the primer with a tail, or about 5× to about 25× the concentration of the primer with a tail, or about 10× to about 20× the concentration of the primer with a tail.

84. The method according to any one of claims 80 to 83, wherein the non-cleavable probe is prepared at a concentration different from the concentration of the primer having the tail and the concentration of the primer without the tail.

85. The method according to claim 84, wherein the non-cleavable probe is prepared at a concentration higher than the concentration of the primer having the tail.

86. The method according to claim 84, wherein the non-cleavable probe is prepared at a concentration lower than the concentration of the primer without the tail.

87. The method according to any one of claims 84 to 86, wherein the non-cleavable probe is prepared at a concentration of about 2× to about 10× the concentration of the primer without the tail, or at a concentration of about 3× to about 7.5× the concentration of the primer without the tail.

88. The reaction mixture A primer pair for targeting a second nucleic acid target different from the nucleic acid target and generating a second amplicon, the primer pair including a primer having a tail and a primer without a tail, prepared at different concentrations, A detectably labeled cleavable probe configured to hybridize to the second amplicon and generate a luminescence signal corresponding to the amount of the generated second amplicon, Further comprising By subjecting the reaction mixture to an amplification process, the second amplicon is generated, and the cleavable probe generates a luminescence signal without being caused by cleavage in the amplification process, The method further includes a step of measuring the luminescence signal from the cleavable probe. The method according to claim 81.

89. A method for detecting the presence or amount of a first target and / or a second target in a reaction mixture, A step of including a first probe and a second probe in the reaction mixture, wherein the first probe includes a first label capable of specifically interacting with a first target and generating a first detectable signal, and the second probe includes a second label capable of specifically interacting with a second target and generating a second detectable signal, A step of specifically interacting the first probe and the second probe with any first target and second target, respectively, in the reaction mixture Measuring a first total signal through an optical filter under a first set of conditions, wherein the first total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and under the first set of conditions, the first detectable signal increases as a result of specific interaction between the first probe and the first target, while the second detectable signal does not increase as a result of specific interaction between the second probe and the second target; Measuring a second total signal through the same optical filter under a second set of conditions, wherein the second total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and under the second set of conditions, the second detectable signal increases as a result of specific interaction between the second probe and the second target; Evaluating the presence or amount of the first target and / or the second target by estimating the first detectable signal and the second detectable signal based on both the first total signal and the second total signal; A method comprising the above steps.

90. The method according to claim 89, wherein the first label and the second label are identical and / or generate substantially the same fluorescence.

91. The method according to claim 89 or 90, wherein the second fluorescence signal differs more significantly between the first set of conditions and the second set of conditions than the first fluorescence signal does between the first set of conditions and the second set of conditions.

92. The method according to any one of claims 89 to 91, wherein the first probe is a cleavable probe.

93. The method according to claim 92, wherein the increase in the first detectable signal indicates that the cleavable probe has been cleaved.

94. The method according to claim 92 or 93, wherein the first probe includes a fluorophore and a quencher, and is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved.

95. The method according to claim 94, wherein the first probe is a TaqMan probe.

96. The method according to any one of claims 89 to 95, wherein the second probe is a non-cleavable probe.

97. The method according to claim 96, wherein the second probe includes a stem-loop portion capable of forming a stem-loop structure when the second probe is single-stranded.

98. The method according to claim 96 or claim 97, further comprising a quencher, wherein the second label of the second probe is a fluorophore, and the fluorophore is quenched when the second probe is single-stranded, but is arranged so as to be capable of emitting light when the second probe is incorporated into a double-stranded nucleic acid.

99. The method according to claim 98, wherein the fluorophore is located at or near the 5'-end of the second probe, and the quencher is on the 3'-side of the fluorophore.

100. The method according to claim 98 or claim 99, wherein both the fluorophore and the quencher are arranged in or near the stem-loop portion of the second probe.

101. The melting temperature (T ｍ ) of the first probe and the T ｍ of the second probe are within about 8 °C of each other, or within about 6 °C of each other, or within about 4 °C of each other, or within about 2 °C of each other. The method according to any one of claims 89 to 100.

102. The method according to any one of claims 89 to 101, wherein the first set of conditions includes a first measurement temperature at which the first fluorescence signal is measured, and the second set of conditions includes a second different measurement temperature at which the second fluorescence signal is measured.

103. The method according to claim 102, wherein the first measurement temperature and the second measurement temperature differ by at least about 10 °C, about 15 °C, about 20 °C, about 25 °C or about 30 °C or more.

104. The method according to claim 102 or claim 103, wherein at least one of the first measurement temperature or the second measurement temperature is a denaturation temperature at which DNA in the reaction mixture denatures, such as about 90 °C or more.

105. The method according to any one of claims 89 to 104, further comprising thermal cycling of the reaction mixture between two target temperatures over a plurality of cycles.

106. The method according to claim 105, wherein the thermal cycling cycles between two target temperatures over at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cycle.

107. The method according to any one of claims 54 to 106, wherein the step of measuring the signal is performed in an endpoint thermal cycle of the amplification process.

108. The method according to any one of claims 1, 2 or 86, wherein the first probe is configured to generate a cumulative signal over different stages of a cycle of the amplification process, and the second probe is configured to generate a transient signal at different stages of a cycle of the amplification process.

109. The second probe is a compound having the formula: 【Chemical 1】 or a salt thereof, wherein Q is an internal quencher moiety having the formula: [Chemical Formula 2] B is a divalent nucleobase, L １ is a divalent linker, L ５ is a divalent oligonucleotide linker containing 4 to 40 nucleotides, L ５０ is a bond, -NH-, -O-, -S-, -S(O)-, -S(O) ２ -, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene, R ５０ is the second label or the detectable label, R ３０ is -OR ３０Ａ and R ３０Ａ is a monovalent oligonucleotide moiety, R ２ is hydrogen or -OR ２Ａ and R ４ is hydrogen or unsubstituted methyl, or R ２ substituent and R ４ substituents are joined to form a substituted or unsubstituted heterocycloalkyl, R １ and R １０ is, independently, hydrogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -OCl, -OCF ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl, R ６ 、 R ７ 、 R ８ and R ９ are, independently, hydrogen, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ R Ａ , -SO ２ NH ２ , □NHNH ２ , □ONH ２ , □NH C(O)NH ２ , -NHSO ２ H, -NH C(O)H, -NH C(O)OH, -NHOH, -OCl ３ , -OBr ３ , -OF ３ , -OCI ３ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ F, -OCH ２ I, -OCHCl ２ , -OCHBr ２ , -OCHF ２ , -OCHI ２ , -SF ５ , -N ３ , a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl, R １ and R ６ may combine to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl. R ８ and R １０ may combine to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl, and R ２Ａ and R Ａ are, independently, hydrogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -OCl, -OCF ３ , -OCF ３ , -OBr, -OCI ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl The method according to any one of claims 1 to 108.

110. When the second probe is single-stranded, L ５ forms a stem-loop structure, the method according to claim 109.

111. R ５０ is a fluorophore, and Q and R ５０ are arranged apart from each other such that R ５０ is quenched when the second probe is single-stranded and is not quenched when the second probe is incorporated into the double-stranded amplicon, the method according to claim 109.

112. Q and R ５０ The method according to claim 111, wherein both are arranged in or near the stem-loop portion of the second probe.

113. L ５ The method according to claim 109, wherein L comprises 11 to 30 nucleotides.

114. L ５ The method according to claim 109, wherein L comprises 19 to 23 nucleotides.

115. L ５ The method according to claim 109, wherein L comprises 4 to 14 nucleotides.

116. L ５ The method according to claim 109, wherein L comprises 6 to 12 nucleotides.

117. The method according to claim 109, wherein the nucleotide is a DNA nucleotide.

118. The method according to claim 109, wherein the nucleotide is an RNA molecule.

119. The compound has the formula: [Chemical Formula 3]

120. The compound has the formula: 【Chemical Formula 4】

121. The compound has the formula: 【Chemical Formula 5】

122. The compound has the formula: [Chemical Formula 6]

123. The method according to claim 109, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, a divalent uracil or a derivative thereof, a divalent hypoxanthine or a derivative thereof, a divalent xanthine or a derivative thereof, a divalent 7-methylguanine or a derivative thereof, a divalent 5,6-dihydrouracil or a derivative thereof, a divalent 5-methylcytosine or a derivative thereof, or a divalent 5-hydroxymethylcytosine or a derivative thereof.

124. The method according to claim 109, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, or a divalent uracil or a derivative thereof.

125. The method according to claim 109, wherein the compound has the following formula: 【Chemical 7】 The method according to claim 109, wherein the compound has the following formula:

126. The method according to claim 109, wherein the compound has the following formula: 【Chemical 8】 The method according to claim 109, wherein the compound has the following formula:

127. The method according to claim 109, wherein the compound has the following formula: 【Chemical Formula 9】 The method according to claim 109, wherein the compound has the following formula:

128. The method according to claim 109, wherein the compound has the following formula: 【Chemical Formula 10】 The method according to claim 109, wherein the compound has the following formula:

129. L １ is L １０１ -L １０２ -L １０３ -L １０４ -L １０５ and L １０１ 、 L １０２ 、 L １０３ 、 L １０４ and L １０５ are each independently a bond, -NH-, -O-, -S-, -S(O)-, -S(O) ２ 2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, the method according to claim 109.

130. L １０１ is - S(O) ２ - as in the method according to claim 129.

131. L １０２ The method according to claim 129, wherein L is an unsubstituted 3- to 8-membered heterocycloalkyl.

132. L １０２ The method according to claim 129, wherein L is unsubstituted piperidinyl.

133. L １０２ is 【Chemical Formula 11】 The method according to claim 129, wherein...

134. L １０３ The method according to claim 129, wherein L is -C(O)NH-.

135. L １０４ The method according to claim 129, wherein L is unsubstituted C1-C10 alkylene, unsubstituted 2-6 membered heteroalkylene or unsubstituted phenylene.

136. L １０４ is unsubstituted n - hexylene, 【Chemical Formula 12】 The method according to claim 129, wherein...

137. L １０５ is unsubstituted C １ -C １０ alkylene, substituted or unsubstituted 2- to 8-membered heteroalkylene, or unsubstituted 5- to 10-membered heteroarylene, the method according to claim 129.

138. L １０５ is 【Chemical 13】 The method according to claim 129, wherein...

139. L １ is 【Chemical 14】 The method according to claim 129, wherein...

140. L ５０ The method according to claim 109, wherein ５０ is a substituted 2- to 10-membered heteroalkylene.

141. L ５０ is 【Chemical 15】 The method according to claim 109, wherein...

142. R ５０ The method according to claim 109, wherein R is a fluorescent moiety.

143. R ５０ The method according to claim 142, wherein R is in the monovalent form of FAM, VIC, ABY, JUN, AF647, Cy5, AF676 or Cy5.

5.

144. R ２ The method according to claim 109, wherein R is hydrogen or -OH.

145. The method according to claim 109, wherein R2 is hydrogen.

146. R ３０ The method according to any one of claims 109 to 145, wherein R is -OH.

147. R ３０ is 【Chemical Formula 16】 The method according to any one of claims 109 to 145, wherein...

148. The 3'-blocking moiety is monovalent dideoxycytidine (3'ddC), monovalent dideoxyadenosine (ddA), 3'-reverse dT, a 3'-amino modifier, monovalent QSY7, monovalent QSY21, monovalent QSY9, monovalent BHQ1, monovalent BHQ2, monovalent BHQ3, monovalent Dabcyl, monovalent Dabsyl, monovalent Eclipse, monovalent BBQ-650, monovalent Iowa Black RQ, monovalent Iowa Black FQ, 【Chemical 17】 The method according to any one of claims 109 to 145, which is as described above.

149. A composition for detecting nucleic acid in a sample, A first probe detectably labeled with a first label configured to generate a first emission signal, A second probe detectably labeled with a second label configured to generate a second emission signal, Comprising The first probe and the second probe have different sequences, The first label and the second label are the same and / or generate substantially the same emission, Under a first set of conditions, the first label generates a first emission signal that increases as a result of specific interaction between the first probe and a first nucleic acid target, and the second label generates a second emission signal that does not increase as a result of specific interaction between the second probe and a second nucleic acid target, Under a second set of conditions different from the first set of conditions, the second emission signal increases as a result of specific interaction between the second probe and the second nucleic acid target, Composition.

150. The composition according to claim 149, wherein the first probe is a cleavable probe.

151. The first probe comprises a fluorophore and a quencher, and the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved in the annealing / extension step of the amplification process. The method according to any one of claims 149 to 150.

152. The composition according to any one of claims 149 to 151, wherein the first probe is a TaqMan probe.

153. The composition according to any one of claims 149 to 152, wherein the second probe is a non-cleavable probe.

154. The composition according to any one of claims 149 to 152, wherein the second probe includes a stem-loop portion configured to form a stem-loop structure when the second probe is single-stranded.

155. The composition according to any one of claims 153 or 154, wherein the second probe is a fluorophore and a quencher, and the fluorophore is quenched when the second probe is single-stranded, and the fluorophore is not quenched when the second probe is incorporated into a double-stranded amplicon, and the fluorophore and the quencher are arranged apart from each other.

156. The composition according to claim 155, wherein the fluorophore is located at or near the 5' end of the second probe, and the quencher is on the 3' side of the fluorophore.

157. The composition according to any one of claims 155 or 156, wherein both the fluorophore and the quencher are arranged in or near the stem-loop portion of the second probe.

158. The composition according to any one of claims 149 to 157, wherein the reaction mixture further comprises a first primer pair complementary to a first nucleic acid target of the nucleic acid or its complement, wherein the first nucleic acid target is configured to generate a first amplicon to which the first probe can hybridize, and a second primer pair complementary to a second nucleic acid target of the nucleic acid or its complement, wherein the second nucleic acid target is configured to generate a second amplicon to which the second probe can hybridize.

159. The composition according to claim 158, wherein the second primer pair includes a primer having a tail.

160. The composition according to claim 159, wherein the tail forms the 5' end of the primer having the tail.

161. The composition according to any one of claims 159 or 160, wherein the second probe can hybridize to the tail or its complement.

162. The composition according to any one of claims 159 to 161, wherein the second primer pair further comprises a primer without a tail, and the concentration of the primer with a tail in the reaction mixture is different from the concentration of the primer without a tail in the reaction mixture.

163. The composition according to claim 162, wherein the concentration of the primer without a tail is higher than the concentration of the primer with a tail.

164. The composition according to claim 163, wherein the concentration of the primer without a tail is about 2× to about 30×, or about 5× to about 25×, or about 10× to about 20× the concentration of the primer with a tail.

165. The composition according to any one of claims 162 to 164, wherein the second probe is prepared at a concentration different from the concentration of the primer with a tail and the concentration of the primer without a tail.

166. The composition according to claim 165, wherein the second probe is prepared at a concentration higher than the concentration of the primer with a tail.

167. The composition according to any one of claims 165 or 166, wherein the second probe is prepared at a concentration lower than the concentration of the primer without a tail.

168. The composition according to any one of claims 165 to 167, wherein the second probe is prepared at a concentration of about 2× to about 10×, or about 3× to about 7.5× the concentration of the primer with a tail.

169. A primer pair complementary to a nucleic acid target or its complement for generating an amplicon, A non-cleavable probe configured to hybridize to the amplicon and configured to produce a luminescence signal corresponding to the amount of the generated amplicon, comprising In an amplification process, the detectable label includes a series of thermal cycling steps including at least two different target temperatures, and generates luminescence even if the non-cleavable probe is not cleaved. Composition.

170. The composition according to claim 169, wherein the composition is a reaction mixture.

171. The composition according to claim 169 or claim 170, comprising a stem-loop portion capable of forming a stem-loop structure when the non-cleavable probe is single-stranded.

172. The method according to any one of claims 159 to 170, wherein the non-cleavable probe is a fluorophore and a quencher, and the fluorophore is quenched when the non-cleavable probe is single-stranded, but emits light when the probe is incorporated into a double-stranded amplicon, and the fluorophore and the quencher are disposed apart from each other.

173. The composition according to claim 172, wherein the fluorophore is located at the 5' end or near the 5' end of the probe, and the quencher is on the 3' side of the fluorophore.

174. The composition according to claim 172, wherein both the fluorophore and the quencher are in or near the stem-loop portion of the probe.

175. The composition according to any one of claims 169 to 174, wherein the primer pair comprises a primer having a tail.

176. The composition according to claim 175, wherein the tail forms the 5' end of the primer having the tail.

177. The composition according to claim 175 or claim 176, wherein the non-cleavable probe is configured to hybridize to the tail or its complement.

178. The composition according to claim 177, wherein the 3' portion of the non-cleavable probe is configured to hybridize to the tail or its complement.

179. The composition according to any one of claims 169, wherein the primer pair comprises a primer having a tail and a primer without a tail prepared at different concentrations.

180. The composition according to claim 179, wherein the primer without a tail is prepared at a higher concentration than the primer having a tail.

181. The composition according to claim 180, wherein the primer without a tail is prepared at a concentration of about 2× to about 30× the concentration of the primer having a tail, or about 5× to about 25× the concentration of the primer having a tail, or about 10× to about 20× the concentration of the primer having a tail.

182. The composition according to claim 179, wherein the concentration of the non-cleavable probe in the reaction mixture is higher than the concentration of the primer having the tail in the reaction mixture.

183. The composition according to claim 179, wherein the non-cleavable probe is prepared at a concentration lower than the concentration of the primer without the tail.

184. The composition according to any one of claims 183, wherein the non-cleavable probe is prepared at a concentration of about 2× to about 10× the concentration of the primer without the tail, or about 3× to about 7.5× the concentration of the primer without the tail.

185. The reaction mixture is a primer pair complementary to a second nucleic acid target or its complement for generating a second amplicon, a cleavable probe configured to hybridize to the second amplicon and comprising a detectable label configured to generate a luminescence signal corresponding to the amount of the generated second amplicon, further comprising by subjecting the reaction mixture to the amplification process, a second amplicon is generated, and in the amplification process, cleavage of the cleavable probe causes the detectable label of the cleavable probe to generate luminescence, the method further comprising the step of measuring the luminescence signal from the cleavable probe, The composition according to any one of claims 169 to 184.

186. A kit comprising the composition according to any one of claims 149 to 185.

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