Nucleic acid modifying reagent and use thereof

Abstract

To provide compounds, compositions, kits, and methods for DNA amplification reactions.SOLUTION: To provide compounds, compositions, and methods for nucleic acid amplification reactions, particularly for viability PCR (vPCR) applications of compounds that selectively bind to nucleic acids from non-living cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 073,787, filed September 2, 2020, which is incorporated herein by reference in its entirety.

[0002] Provided herein are compounds, compositions, kits, and methods for DNA amplification reactions. In particular, the present disclosure provides compounds that can spontaneously modify nucleic acids from non-viable cells, and corresponding methods for selectively detecting nucleic acids from viable cells via amplification, such as polymerase chain reaction (PCR) and isothermal amplification methods. [Background technology]

[0003] Nucleic acid-based analytical methods, ranging from species-specific PCR to metagenomics, have greatly expanded our understanding of microbial diversity in natural samples. However, cell viability cannot be easily assessed by standard DNA-targeted or isothermal amplification methods, such as PCR or qPCR.

[0004] Propidium monoazide (PMA) has been shown to distinguish between DNA associated with viable and nonviable cells in a technique known as viability PCR (vPCR). In samples containing both viable and dead cells, PMA can access DNA from dead cells due to their compromised cell membranes. PMA contains a photoreactive azide moiety that covalently modifies DNA upon photolysis, rendering it unable to serve as a template in the amplification reaction. After the photoactivation step, lysis of remaining viable cells exposes DNA, allowing amplification. However, variations in light spectrum and intensity can lead to sample-to-sample variability, especially in complex and turbid samples. These issues limit the widespread use and adoption of vPCR technology. Summary of the Invention

[0005] Provided herein are compounds, compositions, kits, and methods for DNA amplification reactions. In particular, the present disclosure provides compounds that can spontaneously modify nucleic acids from non-viable cells, and corresponding methods for selectively detecting nucleic acids from viable cells via amplification, such as polymerase chain reaction (PCR) and isothermal amplification methods.

[0006] In one aspect, provided herein is a compound or a salt thereof, the compound comprising: (A) Nucleic acid binding portion (“NAB portion”), (B) a live / dead cell discrimination portion ("LDCD portion"), and (C) Contains a nucleic acid modifying moiety ("NAM moiety").

[0007] In some embodiments, the A compound has the structure: ABC and wherein A is a NAB moiety, B is an LDCD moiety, and C is a nucleic acid-modifying moiety. In some embodiments, the compound has two or more NAB moieties, two or more LDCD moieties, and / or two or more NAM moieties. In some embodiments, the NAB moiety is a groove-binding moiety, an intercalating moiety, or a mixed-mode binding moiety. In some embodiments, the NAB moiety comprises a bibenzimidazole moiety or a phenylphenanthridium moiety. In some embodiments, the NAB moiety is [ka] The compound has a structure selected from:

[0008] In some embodiments, the NAM moiety comprises a bischloroethylamine (nitrogen mustard) moiety, a platinum-based moiety, a 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indolyl moiety, or a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) moiety. [ka] The compound has a structure selected from:

[0009] In some embodiments, the LDCD moiety comprises at least one charged moiety. In some embodiments, the LDCD moiety comprises at least one quaternary ammonium group. In some embodiments, the LDCD moiety comprises at least one poly(ethylene glycol) moiety. In some embodiments, the poly(ethylene glycol) moiety has the formula: -(CH2CHO) n- wherein n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the LDCD moiety comprises a functional group attached to a solid support. In some embodiments, the LDCD moiety comprises a metabolically cleavable group.

[0010] In some embodiments, the compound is [ka] [ka] [ka] [ka] and any salt thereof.

[0011] In one aspect, provided herein is a method for detecting viable microorganisms or living cells in a sample, the method comprising: (a) contacting a sample with a compound disclosed herein (i.e., a compound having a NAB moiety, an LDCD moiety, and a NAM moiety, or a salt thereof) to form a first mixture; (b) contacting the first mixture with an inactivating agent to form a second mixture; and (c) amplifying nucleic acids from the second mixture to produce a detectable signal, the signal indicating the presence of viable microorganisms or living cells in the sample.

[0012] In some embodiments, the method does not include a photoactivation step. In some embodiments, the method does not include a culturing step.

[0013] In some embodiments, step (a) comprises contacting the sample with the compound or its salt for 5 to 180 minutes. In some embodiments, step (a) comprises contacting the sample with the compound or its salt for 60 to 120 minutes. In some embodiments, step (a) comprises adding to the sample a composition comprising the compound or its salt in a solvent. In some embodiments, step (a) further comprises contacting the sample with one or more additional compounds disclosed herein (i.e., a compound having a NAB moiety, an LDCD moiety, and a NAM moiety, or a salt thereof). In some embodiments, the solvent is dimethyl sulfoxide. In some embodiments, in step (a), the first mixture comprises the compound at a concentration of 5 to 100 micromolar.

[0014] In some embodiments, the inactivating agent comprises a nucleophile selected from an amine and a thiol, hi some embodiments, the inactivating agent is selected from cysteine, glutathione, dNTPs, guanine, an amine-containing buffer, and any mixture thereof.

[0015] In some embodiments, step (c) comprises: (i) lysing cells in a second mixture to form a lysed sample; (ii) adding a DNA polymerase and an amplification reagent to the lysed sample to form a mixture; and (iii) subjecting the mixture to a thermal cycling protocol to amplify nucleic acids from the sample.

[0016] In some embodiments, the method further comprises removing contaminants and / or cellular debris from the lysed sample prior to adding the DNA polymerase and amplification reagents. In some embodiments, the method further comprises heating the mixture to a temperature of at least 90°C to activate the DNA polymerase prior to subjecting the mixture to a thermal cycling protocol.

[0017] In some embodiments, the DNA polymerase is a thermostable DNA polymerase selected from Taq, Tbr, Tfi, Tfl, KOD, Tru, Tth, Tli, Tac, Tne, Tma, Pfu, Pho, Pwo, ES4, Bca, Bst, Sac, Sso, Poc, Pab, and Mth, or a mutant, variant, or derivative thereof. In some embodiments, the DNA polymerase is Taq polymerase. In some embodiments, the amplification reagent comprises at least one primer, deoxynucleotide triphosphates, a buffer, and a magnesium salt. In some embodiments, the magnesium salt is magnesium chloride. In some embodiments, the amplification reagent comprises forward and reverse primers for target amplicons in the sample.

[0018] In some embodiments, the thermal cycling protocol comprises: (1) a denaturation step comprising subjecting the mixture to a temperature of 90-96°C; (2) an annealing step comprising subjecting the mixture to a temperature of 45 to 68°C; (3) an extension step comprising subjecting the mixture to a temperature of 50 to 72°C; The series of steps (1) to (3) is repeated 10 or more times in succession. In some embodiments, the series of steps (1) to (3) is repeated 20 or more times in succession.

[0019] In some embodiments, the thermal cycling protocol comprises: (1a) a denaturation step comprising subjecting the mixture to a temperature of 90-96°C; (2a) an annealing / extension step comprising subjecting the mixture to a temperature of 45-70°C; The series of steps (1a) to (2a) is repeated 10 or more times in succession. In some embodiments, the series of steps (1a) to (2a) is repeated 20 or more times in succession.

[0020] In some embodiments, step (c) comprises: (i) lysing cells in a second mixture to form a lysed sample; (ii) adding a DNA polymerase and an amplification reagent to the lysed sample to form a mixture; and (iii) subjecting the mixture to an isothermal amplification protocol to amplify nucleic acids from the sample.

[0021] In some embodiments, the detectable signal is a fluorescent signal.

[0022] In some embodiments, the microorganism is a bacterium. In some embodiments, the bacterium is from a genus selected from Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Salmonella, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia. In some embodiments, the microorganism is a virus. In some embodiments, the virus is from a family selected from Retroviridae, Picornaviridae, Calciviridae, Flaviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae. In some embodiments, the microorganism or cell is a fungus, yeast, mammalian cell, or plant cell.

[0023] In one aspect, provided herein is a method for amplifying nucleic acids from a sample, the method comprising: (a) contacting a sample with a compound disclosed herein (i.e., a compound having a NAB moiety, an LDCD moiety, and a NAM moiety, or a salt thereof) to form a first mixture; (b) contacting the first mixture with an inactivating agent to form a second mixture; and (c) amplifying nucleic acids from the second mixture.

[0024] In some embodiments, the method does not include a photoactivation step. In some embodiments, the method does not include a culturing step.

[0025] In some embodiments, step (a) comprises contacting the sample with the compound or its salt for 5 to 180 minutes. In some embodiments, step (a) comprises contacting the sample with the compound or its salt for 60 to 120 minutes. In some embodiments, step (a) comprises adding to the sample a composition comprising the compound or its salt in a solvent. In some embodiments, the solvent is dimethyl sulfoxide. In some embodiments, in step (a), the first mixture comprises the compound at a concentration of 5 to 100 micromolar. In some embodiments, the inactivating agent comprises a nucleophile selected from an amine and a thiol. In some embodiments, the inactivating agent is selected from cysteine, glutathione, dNTP, guanine, an amine-containing buffer, or any mixture thereof.

[0026] In some embodiments, step (c) comprises: (i) lysing cells in a second mixture to form a lysed sample; (ii) adding a DNA polymerase and an amplification reagent to the lysed sample to form a mixture; and (iii) subjecting the mixture to a thermal cycling protocol to amplify nucleic acids from the sample.

[0027] In some embodiments, the method further comprises removing contaminants and / or cellular debris from the lysed sample prior to adding the DNA polymerase and amplification reagents.

[0028] In some embodiments, the method further comprises heating the mixture to a temperature of at least 90° C. to activate the DNA polymerase prior to subjecting the mixture to the thermal cycling protocol.

[0029] In some embodiments, the DNA polymerase is a thermostable DNA polymerase selected from Taq, Tbr, Tfi, Tfl, KOD, Tru, Tth, Tli, Tac, Tne, Tma, Pfu, Pho, Pwo, ES4, Bca, Bst, Sac, Sso, Poc, Pab, and Mth, or a mutant, variant, or derivative thereof, hi some embodiments, the DNA polymerase is Taq polymerase.

[0030] In some embodiments, the amplification reagents comprise at least one primer, deoxynucleotide triphosphates, a buffer, and a magnesium salt. In some embodiments, the magnesium salt is magnesium chloride. In some embodiments, the amplification reagents comprise forward and reverse primers for target amplicons in the sample.

[0031] In some embodiments, the thermal cycling protocol comprises: (1) a denaturation step comprising subjecting the mixture to a temperature of 90-96°C; (2) an annealing step comprising subjecting the mixture to a temperature of 45 to 68°C; (3) an extension step comprising subjecting the mixture to a temperature of 50 to 72°C; The series of steps (1) to (3) is repeated 10 or more times in succession. In some embodiments, the series of steps (1) to (3) is repeated 20 or more times in succession.

[0032] In some embodiments, the thermal cycling protocol comprises: (1a) a denaturation step comprising subjecting the mixture to a temperature of 90-96°C; (2a) an annealing / extension step comprising subjecting the mixture to a temperature of 45-70°C; The series of steps (1a) to (2a) is repeated 10 or more times in succession. In some embodiments, the series of steps (1a) to (2a) is repeated 20 or more times in succession.

[0033] In some embodiments, step (c) comprises: (i) lysing cells in a second mixture to form a lysed sample; (ii) adding a DNA polymerase and an amplification reagent to the lysed sample to form a mixture; and (iii) subjecting the mixture to an isothermal amplification protocol to amplify nucleic acids from the sample.

[0034] In some embodiments, the sample is suspected of containing an organism or cell selected from bacteria, viruses, yeast, fungi, mammalian cells, or plant cells. In some embodiments, the sample is suspected of containing bacteria. In some embodiments, the bacteria is from a genus selected from Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Salmonella, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia. In some embodiments, the sample is suspected of containing a virus. In some embodiments, the virus is from a family selected from Retroviridae, Picornaviridae, Calciviridae, Flaviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae.

[0035] In one aspect, provided herein are methods for removing nucleic acids from a sample, the methods comprising contacting the sample with a compound disclosed herein (i.e., a compound having a NAB moiety, an LDCD moiety, and a NAM moiety, or a salt thereof). In some embodiments, the compound is immobilized on a solid support.

[0036] In one aspect, provided herein is a system or kit comprising a compound disclosed herein (i.e., a compound having a NAB moiety, an LDCD moiety, and a NAM moiety, or a salt thereof).

[0037] In some embodiments, the system or kit further comprises a DNA polymerase. In some embodiments, the DNA polymerase is a thermostable DNA polymerase selected from Taq, Tbr, Tfi, Tfl, KOD, Tru, Tth, Tli, Tac, Tne, Tma, Pfu, Pho, Pwo, ES4, Bca, Bst, Sac, Sso, Poc, Pab, and Mth, or a mutant, variant, or derivative thereof. In some embodiments, the DNA polymerase is Taq polymerase. In some embodiments, the system or kit further comprises one or more amplification reagents. In some embodiments, the one or more amplification reagents are selected from at least one primer, deoxynucleotide triphosphates, a buffer, and a magnesium salt. In some embodiments, the system or kit further comprises forward and reverse primers for a target nucleic acid sequence of an organism or cell selected from bacteria, viruses, yeast, fungi, mammalian cells, or plant cells. In some embodiments, the organism is a bacterium from a genus selected from Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia.In some embodiments, the organism is a virus from a family selected from Retroviridae, Picornaviridae, Calciviridae, Flaviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae. [Brief explanation of the drawings]

[0038] [Figure 1] A representative viability assay is shown that does not require a photoactivation step. [Figure 2] Data from vPCR reactions performed on live and dead cells with several concentrations of the compound CS0775 are shown. [Figure 3] Data from vPCR reactions performed on representative Gram-negative bacteria in the presence of various concentrations of DMSO and compound CS0775 in two amplicon sizes are shown. [Figure 4] Data from vPCR reactions in representative bacterial strains in the presence of compound CS0775 are shown, using primers optimized for various amplicon sizes. [Figure 5] 1 shows data from vPCR reactions in representative bacterial strains in the presence of various concentrations of the compound CS0775. [Figure 6] 1 shows data from vPCR reactions in representative bacterial strains in the presence of various concentrations of several compounds of the present disclosure. [Figure 7] 1 shows data from vPCR reactions in samples containing various cell concentrations in the presence of compound CS0775. [Figure 8]1 shows data from vPCR reactions in samples with various proportional combinations of live and dead cells in the presence of compound CS0775. [Figure 9] Shown are data from vPCR reactions performed with intact, heat-inactivated virus particles treated with compound CS0775. DETAILED DESCRIPTION OF THE INVENTION

[0039] Provided herein are compounds, compositions, kits, and methods for performing nucleic acid amplification reactions, including viability PCR (vPCR) reactions. The compounds contain a nucleic acid-binding moiety, a nucleic acid-modifying moiety, and a viability-discriminating motif for distinguishing between DNA associated with viable cells and DNA associated with nonviable cells. This method does not require a photoactivation step and minimizes interference from DNA from dead cells, enabling culture-independent DNA detection from viable cells. The compounds, compositions, kits, and methods can simplify the vPCR process and improve the consistency and robustness of molecular detection-based viability tests. The compounds can also be used for other applications, such as methods for removing nucleic acids from samples.

[0040] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0041] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0042] Definitions of certain functional groups and chemical terms are explained in more detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th In addition, general principles of organic chemistry, as well as specific functional groups and reactivities, are described in detail in Sorrell, Organic Chemistry, 2000, Vol. 1, No. 1, pp. 111-115, 1997. nd edition,University Science Books,Sausalito,2006;Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition,John Wiley&Sons,Inc.,New York,2013;Larock,Comprehensive Organic Transformations,3 rd Edition, John Wiley&Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0043] As used herein, the term "acyl" refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, heteroalkyl, or heteroaryl. Examples of acyl include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, and benzoyl.

[0044] The term "alkoxy," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0045] As used herein, the term "alkyl" refers to an alkyl group having 1 to 30 carbon atoms, for example, 1 to 16 carbon atoms (C1 to C 16 alkyl), 1 to 14 carbon atoms (C1-C 14 alkyl), 1 to 12 carbon atoms (C1-C 12 alkyl), 1 to 10 carbon atoms (C1-C 10 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 6 to 20 carbon atoms (C6-C 20 alkyl), or 8 to 14 carbon atoms (C8-C 14 Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-pentyl, hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0046] As used herein, the term "alkylene" refers to an alkylene group having 1 to 10 carbon atoms (C-C 10It refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms (C1-C6 alkylene), for example. Representative examples of alkylene include -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH2CH2CH2CH2CH 2- , -CH2CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2CH2-, and -CH(CH3)CH2CH2CH2CH2-.

[0047] As used herein, "alkenyl" refers to a straight or branched chain hydrocarbon containing 2 to 30 carbon atoms and containing at least one carbon-carbon double bond. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0048] As used herein, "alkynyl" refers to a straight or branched chain hydrocarbon containing 2 to 30 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0049] The term "aryl" as used herein refers to an aromatic carbocyclic ring system having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic), including fused ring systems, and containing no heteroatoms. As used herein, aryl refers to an aromatic ring system having 6 to 20 carbon atoms (C6-C8). 20 aryl), 6 to 14 ring carbon atoms (C6 to C 14 aryl), 6 to 12 ring carbon atoms (C6 to C 12 aryl), or 6 to 10 ring carbon atoms (C6-C 10Representative examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl.

[0050] The term "arylene" as used herein refers to a divalent aryl group. Representative examples of arylene groups include, but are not limited to, phenylene groups (e.g., 1,2-phenylene, 1,3-phenylene, and 1,4-phenylene).

[0051] The term "cycloalkyl," as used herein, refers to a saturated carbocyclic ring system containing 3 to 10 carbon atoms and 0 heteroatoms. Cycloalkyls can be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0052] As used herein, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0053] The term "halogen" or "halo" as used herein means F, Cl, Br, or I.

[0054] As used herein, the term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms) is replaced with a halogen.

[0055] The term "heteroalkyl," as used herein, refers to an alkyl group, as defined herein, in which one or more carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatomic group, such as -NR-, -O-, -S-, -S(O)-, or -S(O)-, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which may be optionally substituted. By way of example, one, two, or three carbon atoms may be independently replaced with the same or different heteroatomic groups. Examples of heteroalkyl groups include, but are not limited to, -OCH, -CHOCH, -SCH, -CHSCH, -NRCH, and -CHNRCH, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may be optionally substituted. Heteroalkyl also includes groups in which an alkyl carbon atom is oxidized (i.e., -C(O)-).

[0056] As used herein, the term "heteroalkylene" refers to an alkylene group, as defined herein, in which one or more carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group, such as -NR-, -O-, -S-, -S(O)-, or -S(O)-, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which may be substituted. By way of example, one, two, or three carbon atoms may be independently replaced with the same or different heteroatom groups. Heteroalkylene also includes groups in which an alkyl carbon atom is oxidized (i.e., -C(O)-). Examples of heteroalkylene groups include, but are not limited to, -CH-O-CH-, -CH-S-CH-, -CH-NR-CH-, -CH-NH-C(O)-CH, etc., as well as polyethylene oxide chains, polypropylene oxide chains, and polyethyleneimine chains.

[0057] The term "heteroaryl," as used herein, refers to an aromatic group having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic) with one or more ring heteroatoms independently selected from O, N, and S. A monocyclic aromatic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from O, N, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, N, and S). A 5-membered monocyclic aromatic ring has two double bonds, and a 6-membered monocyclic aromatic ring has three double bonds. A bicyclic heteroaryl group is exemplified by a monocyclic aryl group, as defined herein, or a monocyclic heteroaryl ring fused to a monocyclic heteroaryl group, as defined herein. A tricyclic heteroaryl group is exemplified by a monocyclic heteroaryl ring fused to two rings independently selected from a monocyclic aryl group, as defined herein, and a monocyclic heteroaryl group, as defined herein. Representative examples of monocyclic heteroaryl include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzodioxolyl, benzofuranyl, benzoxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isolidinonaphdolyl, isobenzolinofuranyl, isobenzofuranyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl.Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl. The monocyclic, bicyclic, and tricyclic heteroaryl are attached to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0058] As used herein, the term "heterocycle" or "heterocyclic ring" refers to a saturated or partially unsaturated non-aromatic cyclic group having one or more ring heteroatoms independently selected from O, N, and S, and means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from O, N, and S. The 7- and 8-membered rings contain zero, one, two, or three double bonds and one, two, or three heteroatoms selected from O, N, and S. Representative examples of monocyclic heterocycles include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperizinyl, and cyclohexyl. These include, but are not limited to, thiazolinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiaridinyl, 1,3-thiazinanyl, thiazolinyl, thiazolinyl, 1,1-dioxide thiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl. A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms.Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. A tricyclic heterocycle is a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocyclic group in which two non-adjacent atoms of the bicycle are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane). The monocyclic, bicyclic, and tricyclic heteroaryls are attached to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0059] As used herein, the term "hydroxy" refers to an --OH group.

[0060] In some cases, the number of carbon atoms in a group (e.g., alkyl, alkoxy, or cycloalkyl) is indicated by the prefix "C x -C y -" where x is the smallest number and y is the largest number of carbon atoms in the group. Thus, for example, "C1-C3-alkyl" refers to an alkyl group containing from 1 to 3 carbon atoms.

[0061] As used herein, the term "substituent" refers to a group substituted on an atom of the indicated group.

[0062] Where a group or moiety can be substituted, the term "substituted" means that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments, 1, 2, or 3; and in other embodiments, 1 or 2) hydrogens on the group designated with "substituted" can be replaced with a selection of the listed designated groups or with suitable groups known to those of ordinary skill in the art (e.g., one or more of the groups listed below), provided that the normal valence of the designated atom is not exceeded. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0063] For the compounds described herein, groups and substituents thereof may be selected according to the allowed valences of atoms and substituents such that selection and substitution thereof results in stable compounds that do not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like.

[0064] Where substituent groups are identified by their conventional chemical formula written from left to right, they optionally encompass the substituents obtained by writing the structure from right to left, e.g., -CHO-, optionally also listed as OCH-, and OC(O)NH-, and also optionally listed as -NHC(O)O-.

[0065] For the recitation of numerical ranges herein, each intervening number therebetween to the same degree of precision is expressly contemplated. For example, for the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0066] As used herein, a "nucleic acid binding moiety" or "NAB moiety" is a moiety that interacts non-covalently with nucleic acids. Non-limiting examples of NAB moieties include major and minor groove binders (which interact with DNA by binding to the major or minor groove of the DNA double helix), intercalators (planar moieties that insert between nucleotide base pairs), and mixed-mode NAB moieties (which include a moiety that intercalates into the DNA double helix and a moiety that protrudes into a groove such as the minor groove).

[0067] As used herein, a "nucleic acid modifying moiety" or "NAM moiety" is a moiety that reacts with at least one nucleotide of a nucleic acid to form a covalent bond (e.g., a covalent bond or a coordinate covalent bond) to the nucleic acid. A NAM moiety can form a covalent bond with one DNA nucleobase or react with two different DNA nucleobases to form a crosslink within the same strand (intrastrand) or between opposite strands (intrastrand).

[0068] As used herein, the term "cell-permeable" refers to a compound or moiety that can effectively pass through the cell membrane of a synthetically permeabilized non-viable cell or viable cell, or the intact membrane of a cell, whether non-viable or viable. As used herein, the term "cell-impermeable" refers to a compound or moiety that cannot effectively pass through the cell membrane of a viable cell that has not been synthetically permeabilized.

[0069] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and can include fluids, solids, tissues, and gases. Biological samples include blood, serum, and the like. A sample can also refer to a cell lysate, which can include cells lysed with a lysing agent, or lysates such as rabbit reticulocyte or wheat germ lysate. A sample can also include cell-free expression systems. Environmental samples include environmental materials such as surface material, soil, water, crystals, and industrial samples. These examples should not be construed as limiting the types of samples applicable to this disclosure.

[0070] As used herein, the term "substantially" means that the recited property, parameter, and / or value need not be achieved exactly, but that deviations or variations, including, for example, tolerances, measurement errors, measurement accuracy limits, and other factors known to those of skill in the art, may occur in an amount that does not eliminate the effect the property is intended to provide. A substantially absent property or characteristic can be within the noise of a significant property, below background, below the detection capability of the assay being used, or a small percentage (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%).

[0071] "Variant" is used herein to refer to a peptide or polypeptide that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. Representative examples of "biological activity" include enzymatic activity, such as DNA polymerase activity. Variant is also used herein to refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. As used herein, a "conservative" amino acid substitution refers to the replacement of an amino acid in a peptide or polypeptide with another amino acid that has similar chemical properties, such as size or charge. For purposes of this disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) alanine (A) and glycine (G), 2) aspartic acid (D) and glutamic acid (E), 3) asparagine (N) and glutamine (Q), 4) arginine (R) and lysine (K), 5) isoleucine (I), leucine (L), methionine (M), and valine (V); 6) phenylalanine (F), tyrosine (Y), and tryptophan (W); 7) serine (S) and threonine (T), 8) Cysteine ​​(C) and methionine (M).

[0072] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein, and nucleic acid chemistry and hybridization described herein is that which is well known and commonly used in the art. The meaning and scope of terms should be clear, except that in the event of any potential ambiguity, definitions provided herein shall take precedence over any dictionary or extrinsic definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0073] 2.Compound The present disclosure includes compounds that can selectively bind and modify nucleic acids from non-viable cells without the need for a photoactivation step. Such compounds can be used in methods for selectively detecting nucleic acids from viable cells and in methods for removing nucleic acids from samples.

[0074] In one aspect, the disclosure provides a compound or salt thereof, the compound comprising: (A) Nucleic acid binding portion (“NAB portion”), (B) a live / dead cell discrimination portion ("LDCD portion"), and (C) Contains a nucleic acid modifying moiety ("NAM moiety").

[0075] In some embodiments, the compound comprises two or more NAB moieties (e.g., two NAB moieties), two or more LDCD moieties (e.g., two LDCD moieties), and / or two or more NAM moieties (e.g., two NAM moieties). In some embodiments, the compound comprises two or more NAB moieties (e.g., two NAB moieties). In some embodiments, the compound comprises two or more NAM moieties (e.g., two NAM moieties).

[0076] In some embodiments, the compound is ABC, BAC, ACB, BCA, and [ka] wherein A is a NAB moiety, B is an LDCD moiety, and C is a NAM moiety. In some embodiments, the compound has the structure ABC, where A is a NAB moiety, B is an LDCD moiety, and C is a NAM moiety. In such embodiments, the NAB moiety A is linked to the LDCD moiety B via a covalent bond, and the NAM moiety C is linked to the LDCD moiety B by another covalent bond.

[0077] The compound includes at least one NAB moiety, which is a moiety that non-covalently interacts with nucleic acids. The NAB moiety efficiently binds to nucleic acid species and facilitates rapid covalent modification of the nucleic acid by the NAM moiety. In some embodiments, the NAB moiety interacts with DNA by binding to the major groove of the DNA double helix. In some embodiments, the NAB moiety interacts with DNA by binding to the minor groove of the DNA double helix. In some embodiments, the NAB moiety is an intercalating moiety that intercalates between nucleotide base pairs, such as an intercalating dye.

[0078] Suitable NAB moieties include acridine, phenanthridine (e.g., phenylphenanthridium), dipyridine, terpyridine, phenanthroline, indole, quinoline, cyanine, quinacrine, benzothiazole, benzimidazole (e.g., bibenzimidazole), pyridocarbazole (e.g., pyridocarbazole dimer), aminoglycoside, and like groups. Additionally, a wide variety of nucleic acid stains are known, any of which (or portions thereof) can be used as the NAB moiety in the compounds described herein. Examples of nucleic acid stains include, for example, ethidium bromide, Hoescht stain, DAPI, and SYBR Green. Additional nucleic acid stains are described in Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies, 11 (2002). thEdition (2010), Chapter 8, which is incorporated herein by reference in its entirety. Other nucleic acid binding dyes are disclosed in U.S. Patent No. 9,206,474, which is incorporated herein by reference in its entirety. Other compounds known to have nucleic acid binding activity include metal intercalators such as ruthenium and rhodium complexes with ligands such as bipyridine, phenanthroline, 4,4-diphenylbipyridine, and derivatives thereof.

[0079] Representative examples of NAB moieties that can be used in the compounds described herein include the following:

[0080] Single-stranded DNA / RNA binding moieties such as: [ka] wherein X is O, NH, or S, m is 0, 1, or 2, and n is 0, 1, 2, or 3 (e.g., m is 1 and n is 1).

[0081] Intercalating parts such as: [ka]

[0082] Minor groove binders such as: [ka] wherein X is O, NH, or S, m is 0, 1, or 2, and n is 0, 1, 2, or 3 (e.g., m is 1 and n is 1).

[0083] Main groove binders such as: [ka] In the formula, R x is -CH2NH2 or H.

[0084] In certain embodiments, the NAB moiety is selected from: [ka]

[0085] The compounds comprise an LDCD moiety, which contains at least one LDCD motif that allows the compound to bind to and modify DNA from dead cells but not from living cells. The LDCD motif either prevents the molecule from entering the living cell or is processed by the living cell so that the compound cannot modify the living cell DNA. The LDCD motif distinguishes the compounds described herein from other compounds that contain NAB and NAM moieties (e.g., anti-cancer or research drugs, which are designed to be cell-permeable to bind to and modify nucleic acids in living cells). In addition to at least one LDCD motif, the LDCD moiety contains an alkylene group (-(CH2) n The alkyl group may also contain other atoms or groups of atoms, including, but not limited to, -), an ether group (-O-), a thioether group (-S-), an amide bond (-C(O)NH-), an ester bond (-C(O)O-), a carbamate bond (-OC(O)NH-), a sulfonamide bond (-S(O)NH-), a phenylene bond (-CH-), and any combination thereof. Additionally, any substitutable atom or group can be substituted with a suitable substituent (e.g., an amide bond can contain the group -C(O)NR-, where R is a suitable substituent, or a phenylene bond can contain one or more substituents on the phenyl group).

[0086] In some embodiments, the LDCD moiety functions as a linker between one or more NAB moieties and one or more NAM moieties.

[0087] In some embodiments, the LDCD moiety comprises at least one LDCD motif selected from a charged moiety, a high molecular weight moiety, an immobilization moiety, and a metabolically cleavable moiety.

[0088] In some embodiments, the LDCD motif is a charged moiety that will prevent the molecule from entering a living cell. In some embodiments, the LDCD moiety comprises at least one charged moiety selected from a carboxylate group, a sulfate group, a sulfonate group, a phosphate group, a phosphonate group, and an ammonium group (e.g., a quaternary ammonium group). In some embodiments, the LDCD moiety comprises two or more charged moieties. For example, in some embodiments, the LDCD moiety comprises two or more carboxylate groups, two or more sulfate groups, two or more sulfonate groups, two or more phosphate groups, two or more phosphonate groups, or two or more ammonium groups (e.g., two or more quaternary ammonium groups). In some embodiments, the LDCD moiety comprises two, three, four, five, six, or more charged moieties. In some embodiments, the LDCD moiety comprises at least one quaternary ammonium center. In some embodiments, the LDCD moiety has the formula -N + In some embodiments, the LDCD moiety comprises at least one group of the formula -(CH)-. In some embodiments, the LDCD moiety comprises at least one carboxylate group. In some embodiments, the LDCD moiety comprises a group of the formula -CHCOO - or -CH2CH2COO - In some embodiments, the LDCD moiety comprises at least one group of the formula -CH2PO3 2 - or CH2CH2PO3 2- It contains at least one group of

[0089] In some embodiments, the LDCD motif is a high molecular weight moiety, e.g., a moiety in which the entire compound has a molecular weight greater than 600 g / mol, e.g., greater than 700 g / mol, greater than 800 g / mol, greater than 900 g / mol, or greater than 1000 g / mol. High molecular weight compounds can reduce mobility and affect cell permeability. The high molecular weight moiety can include any combination of groups such as alkylene, heteroalkylene, arylene, and heteroarylene moieties, provided that the total molecular weight of the LDCD moiety is sufficient to render the compound unable to enter living cells (e.g., the entire compound has a molecular weight of 600 g / mol or greater). In some embodiments, the high molecular weight moiety is a polyethylene glycol chain (i.e., a group of the formula -(CH2CHO) n - group, where n is an integer between 1 and 100, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100. In some embodiments, the LDCD moiety is a peptide, such as a peptide that has the property of rendering the molecule cell-impermeable and not interfering with DNA binding.

[0090] In some embodiments, the LDCD motif is a metabolically cleavable moiety. While molecules bearing such a moiety may be able to enter both living and non-living cells, living cells will process the moiety to release the NAM moiety, thus preventing covalent modification. Because non-living cells cannot process the moiety, the NAM moiety can effectively interact with nucleic acids and prevent their amplification. For example, a trimethylquinone lock is a metabolically cleavable moiety that, when incorporated into a compound at the appropriate position, is processed in living cells to release the NAB or NAM moiety, preventing the compound from targeting and modifying DNA. For example, in some embodiments, in living cells, the trimethylquinone moiety is reduced by an intracellular reductase enzyme. The resulting trimethylhydroquinone moiety undergoes a lactonization reaction to produce dihydrocoumarin. Further rearrangement of the remainder of the molecule then results in the release of the NAM and / or NAB moieties. See, for example, WO2015 / 116867 and Mustafa et al. Bioconjugate Chem. 2016, 27, 1, 87-101, each of which is incorporated by reference in its entirety. An exemplary reaction is shown in Scheme 1 below.

[0091] Scheme 1. Metabolic cleavage of compounds containing the trimethylquinone-locked LDCD motif [ka]

[0092] In some embodiments, the LDCD motif comprises a solid support. Attachment of the compound to the solid support allows a sample containing viable and non-viable cells to be mixed with the solid support, resulting in DNA from the non-viable cells binding to the solid support, but not from the viable cells. Separating the solid support from the remainder of the sample yields a sample containing only viable cells, which can be used in subsequent amplification reactions. This embodiment also allows for the removal of free DNA from other types of samples, which can be used for applications requiring the generation of DNA-free reagents and / or solutions. The solid support can be, for example, a surface such as beads, resins, magnetic particles, membranes, gels, ionic liquids (see, e.g., Egorova et al. Chem. Rev. 2017, 117, 10, 7132-7189), or the surface of a tube, vial, slide, microtiter plate, cuvette, etc. Methods for immobilizing compounds on solid supports are known to those skilled in the art.

[0093] The compound also contains at least one NAM moiety that functions to covalently modify a nucleic acid, thereby preventing it from being amplified. Exemplary NAM moieties include nitrogen mustards (e.g., bendamustine, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, melphalan, and uramustine), nitrosoureas (e.g., carmustine, chlorozotocin, ethylnitrosourea, fotemustine, lomustine, nimustine, ranimustine, semustine, and streptozocin), and alkylsulfonates (e.g., busulfan). and DNA alkylating agents such as eco-CBI compounds (e.g., 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz(e)indole) and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) compounds (e.g., 8-hydroxy-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5-one). Any of these, or derivatives thereof, can be used as the basis for the NAM moiety in the compounds described herein.

[0094] Other NAM moieties include platinum-based moieties that modify DNA (usually via the N7 position of a guanine residue) through the binding of DNA nucleobases (via coordinate covalent bonds) to the platinum center. Examples of platinum-based chemotherapeutic agents include cisplatin, carboplatin, nedaplatin, ormaplatin, oxaliplatin, phenanthriplatin, picoplatin, and satraplatin, any of which (or derivatives thereof) can be used as the basis for the NAM moiety in the compounds described herein. For example, the compounds can be characterized by monodentate or bidentate ligands (e.g., moieties with one or more primary and / or secondary amines and / or another source of coordinated nitrogen atoms, such as pyridine, quinoline, or phenanthridine moieties) that bind to the platinum center.

[0095] In some embodiments, the NAM moiety is [ka] The group includes a group selected from:

[0096] In some embodiments, the compound is [ka] [ka] [ka] [ka] or any salt thereof.

[0097] The compound may be in the form of a salt. In some embodiments, the neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salt is identical to the parent form of the compound for purposes of this disclosure.

[0098] In particular, if the compound has a functional group that is anionic or potentially anionic (e.g., -COOH becomes -COO - -SO3H can be -SO3 - or -P(O)(OH)2 can be -PO3 2- (which may be , and salts may be formed with one or more suitable cations. Examples of suitable inorganic cations include Li + , Na + , and K + Alkali metal cations such as Ca 2+ and Mg 2+ Examples of suitable organic cations include, but are not limited to, alkaline earth cations such as NH4, ... + ) and substituted ammonium ions (e.g., NH3R1 + , NH2R2 + , NHR3 + , and NR4 + ) Examples of some suitable substituted ammonium ions include, but are not limited to, ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. In some embodiments, the compound is a sodium salt.

[0099] If the compound is cationic or has a functional group that can be cationic (e.g., -NH2 is -NH3 +(which may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, tetrafluoroboric acid, toluenesulfonic acid, trifluoromethanesulfonic acid, and valeric acid. In some embodiments, the compound is a halide salt, such as a chloro, bromo, or iodo salt, hi some embodiments, the compound is a tetrafluoroborate or trifluoromethanesulfonate salt.

[0100] The compounds can be prepared by various methods, including those shown in the examples. The compounds and intermediates herein can be isolated and purified by methods well known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, recrystallization at high or low temperatures, optionally with pretreatment with activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described, for example, in "Vogel's Textbook of Practical Organic Chemistry," 5th edition (1989), by Furniss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM20 2JE, England.

[0101] The reaction conditions and reaction times for each individual step may vary depending on the specific reactants used and the substituents present in the reactants. Specific procedures are described in the Examples section. The reactions can be worked up in conventional ways, for example, by removing the solvent from the residue, and further purified according to methodologies commonly known in the art, such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise noted, starting materials and reagents are commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. If the starting materials are not commercially available, they can be prepared by standard organic chemistry techniques, techniques analogous to known syntheses, structurally similar compounds, or procedures similar to those described in the above schemes or synthetic examples sections.

[0102] Routine experimentation, including appropriate manipulation of the reaction conditions, reagents, and sequence of the synthetic route, protection of chemical functional groups incompatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the method, is within the scope of the invention. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those of skill in the art, and examples thereof can be found in the article by PGM Wuts entitled "Greene's Protective Groups in Organic Synthesis" (5th ed.), John Wiley & Sons, Inc. (2014), which is incorporated herein by reference in its entirety. Synthesis of the compounds of the present invention can be achieved by methods similar to those described in the synthetic schemes and specific examples above.

[0103] If an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using optically active starting materials (prepared, for example, by asymmetric induction in an appropriate reaction step), or by resolving a stereoisomeric mixture of the compound or intermediate using standard procedures (chromatographic separation, recrystallization, enzymatic resolution, etc.).

[0104] Similarly, if a pure geometric isomer of a compound is required, it can be obtained by carrying out any of the procedures described above using the pure geometric isomer as the starting material, or by resolving a mixture of geometric isomers of the compound or intermediate using standard procedures such as chromatographic separation.

[0105] The synthetic schemes and specific examples described are illustrative and should not be construed as limiting the scope of the invention, which is defined in the claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are included within the scope of the claims. 3. Compositions and Systems / Kits

[0106] In some embodiments, the present disclosure provides compositions and systems / kits that include the nucleic acid modifying compounds described herein (i.e., compounds that include a NAB portion, an LDCD portion, and a NAM portion).

[0107] Compositions containing nucleic acid-modifying compounds described herein can further comprise a solvent. In some embodiments, the solvent is water. In such embodiments, the composition can further comprise one or more water-soluble components, such as a salt or a buffer. In some embodiments, the solvent is an organic solvent, such as dimethyl sulfoxide (DMSO). Using DMSO as a solvent for the compounds may provide an additional advantage when amplifying DNA from samples containing Gram-negative bacteria, because such bacteria have an additional lipopolysaccharide layer that is more difficult for the compounds to cross, especially if the cells are non-viable. Using DMSO as a solvent can increase the DNA modification efficiency of samples containing Gram-negative bacteria.

[0108] The present disclosure further provides a system or kit comprising a compound described herein (i.e., a compound comprising a NAB moiety, an LDCD moiety, and a NAM moiety). The system or kit comprises the compound alone or in a solvent such as water or DMSO. When the compound is provided alone, the system or kit may further comprise a solvent in which the compound can be dissolved. The system or kit may further comprise one or more reagents used to perform an amplification reaction, such as a viability PCR reaction.

[0109] In some embodiments, the system or kit further comprises a DNA polymerase.The DNA polymerase that can be used according to these embodiments includes, but is not limited to, any polymerase that can replicate DNA molecules.In some embodiments, the DNA polymerase is a thermostable polymerase that is particularly useful in PCR applications. Thermostable polymerases include those from Thermus aquaticus (Taq), Thermus brockianus (Tbr), Thermus filiformis (Tfi), Thermus flavus (Tfl), Thermococcus kodakaraenis (KOD), Thermus ruber (Tru), Thermus thermophilus (Tth), Thermococcus litoralis (Tli) and other species of the genus Thermococcus, Thermoplasma acidophilum (Tac), Thermotoga neapolitana (Tne), Thermotoga maritima (Tma) and other species of the genus Thermotoga, Pyrococcus furiosus (Pfu), Pyrococcus horikossii (Pho), Pyrococcus woesei (Pwo), Pyrococcus strain ES4 (ES4) and other species of the genus Pyrococcus, Bacillus It has been isolated from a variety of thermophilic bacteria, such as Bacillus caldophilus (Bca), Bacillus sterothermophilus (Bst), Sulfolobus acidocaldarius (Sac), Sulfolobus solfataricus (Sso), Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), and Methanobacterium thermoautotrophicum (Mth), as well as their mutants, variants, or derivatives.Thus, in some embodiments, the system or kit comprises a thermostable DNA polymerase selected from Taq, Tbr, Tfi, Tfl, KOD, Tru, Tth, Tli, Tac, Tne, Tma, Pfu, Pho, Pwo, ES4, Bca, Bst, Sac, Sso, Poc, Pab, and Mth, or mutants, variants, or derivatives thereof. In certain embodiments, the DNA polymerase is Taq polymerase. In other embodiments, the DNA polymerase is a polymerase with strand displacement activity, which is particularly useful in isothermal amplification reactions. DNA polymerases with strand displacement activity are isolated from various organisms, such as Bacillus smithii (Bsm), Bacillus stearothermophilus (Bst), Bacillus subtilis (Bsu), and Bacillus subtilis phage phi29 (phi29), as well as mutants, variants, or derivatives thereof. Thus, in some embodiments, the system or kit comprises a DNA polymerase selected from Bsm, Bst, Bsu, and phi29, or a mutant, variant, or derivative of any of them.

[0110] In some embodiments, DNA polymerases that can be used in accordance with these embodiments include, but are not limited to, commercially available DNA polymerases (e.g., Boehringer Mannheim Corp., Indianapolis, IN; Life Technologies, Inc., Rockville, MD; MilliporeSigma, St. Louis, MO; New England Biolabs, Inc., Beverley, MA; Perkin Elmer Corp., Norwalk, CT; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ; Promega Corporation, Madison, WI; Qiagen, Inc., Valencia, CA; and Stratagene, La Jolla, CA).

[0111] In some embodiments, the system or kit further comprises one or more additional reagents such as at least one primer or at least one pair of primers for amplification of a nucleic acid target, at least one probe and / or dye that allows for detection of amplification, a buffer, a ligase, a reverse transcriptase, a detergent (e.g., a non-ionic detergent), nucleotides (dNTPs and / or NTPs), a magnesium salt (e.g., magnesium chloride), or any combination thereof, among other amplification reagents that would be recognized by one of skill in the art based on this disclosure.

[0112] In some embodiments, the system or kit includes one or more primers. Generally, a primer is a short nucleic acid complementary to a longer template. During replication, the primer can be extended based on the template sequence to generate a longer nucleic acid that is a complementary copy of the template. Extension can occur, inter alia, by sequential addition of individual nucleotides (e.g., by the action of a polymerase) or by joining blocks of nucleotides (e.g., by the action of a ligase linking a pair of primers). Primers can be DNA, RNA, analogs thereof (e.g., artificial nucleic acids), or any combination thereof. Primers can have any suitable length, such as at least about 10 to about 30 nucleotides, or about 15 to about 30 nucleotides, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Primers are typically chemically synthesized. Primers can be provided as at least one pair of primers for amplification of at least one nucleic acid target. The pair of primers can be a forward primer and a reverse primer (ie, a sense primer and an antisense primer) that collectively define the opposing ends (and therefore the length) of the resulting amplicon.

[0113] In some embodiments, the system or kit provides a pair of primers that specifically detect a microorganism or cell of interest. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacterium is from a genus selected from Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia. In some embodiments, the microorganism is a virus. In some embodiments, the virus is a member of the Retroviridae family (e.g., human immunodeficiency viruses such as HIV-1 (also known as HTLV-III, LAV, or HTLV-III / LAV, or HIV-III) and other isolates such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses);Bungaviridae (e.g., Hantaanvirus, Bungavirus, Phlebovirus, Nairovirus, etc.); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV)-1 and and HSV-2, varicella-zoster virus, cytomegalovirus (CMV), herpesviruses; Poxyiridae (smallpox virus, vaccinia virus, poxvirus); Iridoviridae (such as African swine fever virus); and unclassified viruses (e.g., pathogens of spongiform encephalopathies, pathogens of hepatitis delta (thought to be defective satellites of hepatitis B virus), pathogens of non-A, non-B hepatitis (Class 1 = internal infection; Class 2 = parenteral infection (such as hepatitis C); Norwalk and related viruses, and astroviruses)). In some embodiments, the microorganism is a fungus. In some embodiments, the microorganism is a yeast, such as Saccharomyces (e.g., Saccharomyces cerevisiae) or Candida (e.g., Candida). In some embodiments, the cell of interest is a eukaryotic cell, such as a mammalian cell or a plant cell.

[0114] A pair of primers can be designed to detect an amplicon of a specific length. For example, the amplicon can be about 200 base pairs to about 1000 base pairs, or about 400 base pairs to about 750 base pairs. For example, in some embodiments, the primers in the system or kit are for an amplicon of at least about 200 base pairs, at least about 300 base pairs, at least about 400 base pairs, at least about 500 base pairs, at least about 600 base pairs, at least about 700 base pairs, at least about 800 base pairs, at least about 900 base pairs, or at least about 1000 base pairs. In some embodiments, the primers in the system or kit are for an amplicon of about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 base pairs.

[0115] In some embodiments, the system or kit can also include one or more probes, or any nucleic acid bound to at least one label, such as at least one dye. The probe can be a sequence-specific binding partner for a nucleic acid target and / or amplicon. The probe can be designed to enable detection of target amplification based on fluorescence resonance energy transfer (FRET), including one or more nucleic acids linked to a pair of dyes that collectively exhibit fluorescence resonance energy transfer (FRET) when brought into close proximity with each other. The pair of dyes can provide, among other things, a first and second emitter, or an emitter and quencher. Fluorescence emission from the pair of dyes changes when the dyes are separated from each other, such as by cleavage of the probe during primer extension (e.g., a 5' nuclease assay using a TAQMAN probe, etc.), or when the probe hybridizes to an amplicon (e.g., a molecular beacon probe). The nucleic acid portion of the probe can have any suitable structure or origin; for example, it can be a locked nucleic acid, a member of a universal probe library, etc. In other cases, the probe and one of the pair of primers can be combined within the same molecule. For example, a primer-probe molecule may contain a primer sequence at its 3' end and a molecular beacon-type probe at its 5' end. This configuration allows related primer-probe molecules labeled with different dyes to be used in a multiplex assay with the same reverse primer to quantify target sequences that differ by a single nucleotide (single nucleotide polymorphism (SNP)).

[0116] In some embodiments, the system or kit can also include one or more label or reporter molecules. Exemplary dyes used for labeling are fluorescent dyes (fluorophores) and fluorescence quenchers. Reporters include any compound or set of compounds that report a state, such as the extent of a reaction. Exemplary reporters include at least one dye, such as a fluorescent dye or energy transfer pair, and / or at least one oligonucleotide. Exemplary reporters for nucleic acid amplification assays can include probes and / or intercalating dyes (e.g., SYBR Green, ethidium bromide, etc.).

[0117] In some embodiments, the system or kit further comprises a magnesium salt, such as magnesium chloride or magnesium sulfate. In some embodiments, the system or kit further comprises magnesium chloride.

[0118] In some embodiments, the system or kit further includes a buffer. The buffer may be provided as a separate component, or one or more of the other system or kit components (e.g., DNA polymerase) may be provided as a solution in the buffer. Exemplary buffers include tris(hydroxymethyl)aminomethane (Tris) buffer. The buffer may further include a salt, such as an ammonium salt (e.g., ammonium chloride or ammonium sulfate) and / or a potassium salt (e.g., potassium chloride). The buffer may be provided at an appropriate pH, which may be tailored specifically for the DNA polymerase provided with the system or kit. In some embodiments, the buffer has a pH of about 7.5 to about 10, or about 8.0 to about 9.5.

[0119] In some embodiments, the system or kit further comprises a reverse transcriptase, which is used in an RT-PCR reaction to generate complementary DNA (cDNA) from RNA, which can then be amplified in an amplification reaction. RT-PCR reactions can be used to detect various RNA species, such as viral RNA. The system or kit may also comprise other components for RT-PCR, such as a ribonuclease inhibitor to inhibit target degradation during cDNA synthesis.

[0120] In some embodiments, kits are provided that include one or more or all of the components necessary, sufficient, or useful for performing the methods described herein (e.g., compounds described herein, DNA polymerase, and amplification reagents). In some embodiments, the kits include positive control reagents, negative control reagents, quantitative standard reagents, and internal amplification control reagents. In some embodiments, the kits include instructions, which may be written instructions or embodied in a computer-readable medium. The reagents in the kit may be contained in one or more containers (e.g., tubes), and the collection of kit components may be packaged in one or more boxes or other containers that facilitate transportation and storage of the kit.

[0121] 4.How to use Embodiments of the present disclosure include methods for amplifying DNA from a sample, wherein DNA is selectively amplified from viable cells and not from non-viable cells in the sample (i.e., an amplification reaction such as a viability PCR reaction).

[0122] Generally, an amplification reaction involves the process of replication or the formation of copies (e.g., direct copies and / or complementary copies) of a nucleic acid or a segment thereof. Replication reactions generally involve enzymes such as polymerase and / or ligase, among others. The nucleic acid and / or segment being replicated is the template (and / or target) for replication. The reaction also generally involves the process of amplification, or a reaction in which replication occurs repeatedly over time to form multiple copies of at least one segment of the template molecule. Amplification can produce an exponential or linear increase in copy number as amplification progresses. Typical amplifications result in a 1,000-fold or greater increase in copy number and / or signal. Exemplary amplification reactions for the assays disclosed herein can include polymerase chain reaction (PCR) or ligase chain reaction (LCR), each of which is driven by thermal cycling. Thermal cycling generally involves cycles of heating and cooling a reaction mixture to perform successive rounds of denaturation (melting), annealing, and extension. Other exemplary amplification reactions include isothermal amplification methods that use enzymes with strand displacement activity (eg, DNA polymerases).

[0123] In some embodiments, the present disclosure provides a method for detecting viable microorganisms or cells of interest in a sample, the method comprising: (a) contacting a sample with a compound described herein (i.e., a compound comprising a NAB moiety, an LDCD moiety, and a NAM moiety, or a salt thereof) to form a first mixture; (b) contacting the first mixture with an inactivating agent to form a second mixture; and (c) amplifying nucleic acids from the second mixture to produce a detectable signal, the signal indicating the presence of viable organisms in the sample.

[0124] In some embodiments, the present disclosure provides a method of amplifying nucleic acids from a sample, the method comprising: (a) contacting a sample with a compound described herein (i.e., a compound comprising a NAB moiety, an LDCD moiety, and a NAM moiety, or a salt thereof) to form a first mixture; (b) contacting the first mixture with an inactivating agent to form a second mixture; and (c) amplifying nucleic acids from the second mixture.

[0125] In some embodiments, the present disclosure provides a method for detecting viable microorganisms or cells of interest in a sample, the method comprising: (a) contacting a sample with a compound described herein (i.e., a compound comprising a NAB moiety, an LDCD moiety, and a NAM moiety, or a salt thereof) to form a first mixture; (b) removing a compound described herein (i.e., a compound comprising a NAB moiety, an LDCD moiety, and a NAM moiety) from the first mixture to form a second mixture; and (c) amplifying nucleic acids from the second mixture to produce a detectable signal, the signal indicating the presence of viable organisms in the sample.

[0126] As discussed herein, the compounds disclosed herein do not require a photoactivation step to modify nucleic acids (e.g., DNA). This provides an advantage to the viability PCR process, particularly for complex or turbid samples, because it can reduce sample-to-sample variation. Thus, in some embodiments, the methods described herein do not include a photoactivation step.

[0127] Furthermore, the compounds described herein can modify DNA from non-viable cells in a sample, while the LDCD moiety prevents their entry into viable cells. This allows for selective labeling of DNA from non-viable cells in a sample without the need for a culture step to increase the number of viable cells in the sample. Thus, in some embodiments, the methods described herein do not include a culture step.

[0128] The sample in which the microorganism or cell is detected and / or the nucleic acid is amplified can be any sample in which it is desirable to selectively amplify nucleic acids from viable cells or to detect viable microorganisms or cells in the sample. In some embodiments, the sample can be obtained from a human subject. For example, non-limiting examples of samples obtained from a subject can include skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric juice, digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluid from tumor tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, nails, plasma, nasal swab or nasopharyngeal washing, spinal fluid, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, umbilical cord blood, sputum, pus, microbiota, meconium, breast milk, and / or other excrement or biological tissue. The sample can also be processed, extracted, or fractionated from any of the foregoing. In other embodiments, the sample is an environmental sample, such as a sample collected from a natural environment (e.g., soil, a body of water, or outdoor air) or an artificial environment (e.g., a clean room, a hospital facility, a food manufacturing facility, a laboratory facility, a pharmaceutical facility, a spa, a cooling tower, or an air handling system). In some embodiments, the sample is a food, pharmaceutical, water sample, or soil sample.

[0129] In some embodiments, the sample may be one suspected of containing viable microorganisms and in which it is desirable to detect the presence of such viable microorganisms. For example, in some embodiments, the sample is suspected of containing bacteria, such as bacteria from a genus selected from Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia. For example, in certain embodiments, the bacterium is Escherichia coli (e.g., Escherichia coli O157:H7), Legionella pneumophila, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Salmonella enterica, or Staphylococcus aureus (e.g., methicillin-resistant Staphylococcus aureus).In some embodiments, the sample is suspected of containing a virus, such as a virus from a family selected from Retroviridae, Picornaviridae, Calciviridae, Flaviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae. In some embodiments, the sample is suspected of containing a fungus, yeast, or another type of eukaryotic cell, such as a mammalian cell or a plant cell.

[0130] The methods disclosed herein include contacting a sample with a compound comprising a NAB moiety, an LDCD moiety, and a NAM moiety to form a first mixture. The contacting step can be performed by adding the compound to the sample and incubating the sample for a time sufficient for the compound to bind to and modify nucleic acids in the sample that are not present in viable cells (i.e., free nucleic acids or nucleic acids in non-viable cells). The contacting step can be performed for about 5 minutes to about 180 minutes, or about 60 minutes to about 120 minutes, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes. In some embodiments, step (a) comprises contacting the sample with the compound for about 90 minutes.

[0131] The concentration of the compound in the first mixture can range from about 1 micromolar to about 200 micromolar, or from about 5 micromolar to about 100 micromolar, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 micromolar.

[0132] Step (a) can be carried out at any suitable temperature, for example, at room temperature (e.g., about 20-25°C) or about 37°C.

[0133] In some embodiments, the contacting step of step (a) is performed by adding a composition comprising the compound, e.g., a composition comprising the compound and a solvent, to the sample. In some embodiments, the solvent is water. In some embodiments, the solvent is an organic solvent such as dimethyl sulfoxide (DMSO). As noted above, the use of a solvent such as DMSO can be particularly advantageous in samples containing Gram-negative bacteria.

[0134] Step (b) of the method involves removing, inactivating, or neutralizing compounds in the first mixture to form a second mixture. In some embodiments, step (b) involves contacting the first mixture with an inactivating agent to form a second mixture. This step serves to inactivate NAM moieties contained in excess compounds in the first mixture, thereby preventing them from further modifying nucleic acids and ensuring optimal performance in subsequent amplification reactions. For example, if excess compounds remain after step (a), subsequent lysis of live cells in the sample may be altered by the excess compounds, making them unavailable for amplification reactions. In such embodiments, the inactivating agent will depend on the particular NAM moiety in the compound. For example, if the NAM moiety is a nitrogen mustard moiety, the inactivating agent can be a nucleophilic compound that can effectively replace chloride groups, such as amines or thiols. Thus, in some embodiments, the inactivating agent is a thiol-containing compound, such as cysteine, glutathione, or DTT. In some embodiments, the inactivating agent is an amine-containing compound, such as an amine-containing buffer. Examples of amine-containing buffers include tris(hydroxymethyl)aminomethane (Tris) and triethanolamine-containing buffers. In some embodiments, the inactivating agent is a dNTP or a mixture of dNTPs. In some embodiments, the inactivating agent is a nucleotide such as guanine. In other embodiments, the inactivation step serves to inhibit the NAB moiety, thereby preventing the compound from binding to and modifying DNA. In such embodiments, the inactivating agent depends on the particular NAB moiety in the compound; in still other embodiments, the compound is physically removed from the first mixture to form a second mixture, for example, by washing the sample.

[0135] Step (c) comprises amplifying nucleic acids from the second mixture to produce a detectable signal, the signal indicating the presence of viable organisms in the sample. The amplification reaction can be carried out in a variety of ways. For example, in some embodiments, the amplification step (c) comprises (i) lysing cells in the second mixture to form a lysed sample, (ii) adding a DNA polymerase and amplification reagents to the lysed sample to form a mixture, and (iii) subjecting the mixture to a thermal cycling protocol to amplify nucleic acids from the sample. For example, in some embodiments, the amplification step (c) comprises (i) lysing cells in the second mixture to form a lysed sample, (ii) adding a DNA polymerase and amplification reagents to the lysed sample to form a mixture, and (iii) subjecting the mixture to an isothermal amplification reaction to amplify nucleic acids from the sample.

[0136] The lysis step exposes DNA from live cells in the sample so that it can be amplified in an amplification reaction. Any suitable method of cell lysis, such as chemical lysis, electrochemical lysis, acoustic lysis (i.e., sonication), mechanical lysis, or thermal lysis, can be used in the method.

[0137] In some embodiments, the method further comprises removing contaminants and / or cellular debris from the lysed sample prior to adding the DNA polymerase and amplification reagents. For example, this step may comprise purifying DNA from the sample to produce a purified DNA sample to which the DNA polymerase and amplification reagents can be added. The DNA may be purified by any conventional means, for example, using organic extraction followed by ethanol precipitation, salt-based precipitation, magnetic particle-based isolation, or solid-phase adsorption.

[0138] DNA is amplified in an amplification reaction such as PCR. Generally, PCR includes any nucleic acid amplification reaction that relies on alternating cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed by thermal cycling between two or more temperature set points, such as a higher melting (denaturing) temperature and a lower annealing / extension temperature, or between three or more temperature set points, such as a higher melting temperature, a lower annealing temperature, and an intermediate extension temperature, among others. PCR generally exponentially increases the amount of product amplicon with successive cycles.

[0139] Any suitable PCR methodology or combination of methodologies may be utilized in the embodiments disclosed herein, such as allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, end-point PCR, hot-start PCR, in situ PCR, inter-sequence specific PCR, inverse PCR, linear after exponential PCR, ligation-mediated PCR, methylation-specific PCR, miniprimer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nested PCR, overlap extension PCR, polymerase cycling assembly, qualitative PCR, quantitative PCR, real-time PCR, RT-PCR, single-cell PCR, solid-phase PCR, thermal asymmetric interlaced PCR, touchdown PCR, universal fast walking PCR, or any combination thereof, among others.

[0140] In some embodiments, DNA is amplified in an isothermal amplification reaction, which generally includes any nucleic acid amplification reaction that relies on enzymes rather than heat denaturation to directly unwind the DNA double helix to synthesize a complementary strand. Examples of isothermal amplification methods include loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HAD), rolling circle amplification (RCA), multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), whole genome amplification (WGA), and recombinase polymerase amplification (RPA).

[0141] Amplification can be performed by adding amplification reagents and a DNA polymerase to form a mixture. DNA polymerases that can be used in accordance with these embodiments include, but are not limited to, any polymerase capable of replicating DNA molecules. In some embodiments, the DNA polymerase is a thermostable polymerase, particularly useful in PCR applications. In some embodiments, the thermostable DNA polymerase is selected from Taq, Tbr, Tfi, Tfl, KOD, Tru, Tth, Tli, Tac, Tne, Tma, Pfu, Pho, Pwo, ES4, Bca, Bst, Sac, Sso, Poc, Pab, and Mth, or mutants, variants, or derivatives thereof. In some embodiments, the DNA polymerase is Taq polymerase. In some embodiments, the DNA polymerase is a polymerase with strand displacement activity useful in isothermal amplification applications. In some embodiments, the DNA polymerase with strand displacement activity is selected from Bsm, Bst, Bsu, and phi29 DNA polymerase.

[0142] The one or more additional amplification reagents may be selected from at least one primer or at least one pair of primers for amplification of a nucleic acid target, at least one probe and / or dye that allows for detection of amplification, a buffer, a ligase, a reverse transcriptase, a detergent (e.g., a non-ionic detergent), nucleotides (dNTPs and / or NTPs), a magnesium salt (e.g., magnesium chloride), or any combination thereof, among other amplification reagents that will be recognized by those of skill in the art based on this disclosure. For example, in some embodiments, the amplification reagents include at least one primer, deoxynucleotide triphosphates (dNTPs), a buffer, and a magnesium salt.

[0143] In some embodiments, the amplification reagents include one or more primers. The primers can be DNA, RNA, analogs thereof (e.g., artificial nucleic acids), or any combination thereof. The primers can have any suitable length, such as at least about 10 to about 30 nucleotides, or about 15 to about 30 nucleotides, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Primers are typically chemically synthesized. Primers can be provided as at least one pair of primers for amplification of at least one nucleic acid target. The pair of primers can be a forward primer and a reverse primer (i.e., a sense primer and an antisense primer), which collectively define the ends (and therefore the length) of the resulting amplicon.

[0144] In some embodiments, the amplification reagents comprise a pair of primers that specifically detect an organism or cell type of interest. In some embodiments, the organism is a microorganism. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacterium is from a genus selected from Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia. In some embodiments, the microorganism is a virus. In some embodiments, the virus is from a family selected from Retroviridae, Picornaviridae, Calciviridae, Flaviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae. In some embodiments, the microorganism is a fungus. In some embodiments, the microorganism is a yeast. In some embodiments, the cell type of interest is a plant cell or a mammalian cell.

[0145] A pair of primers can be designed to detect an amplicon of a specific length. For example, the amplicon can be about 200 base pairs to about 1000 base pairs, or about 400 base pairs to about 750 base pairs. For example, in some embodiments, the primers are for an amplicon of at least about 200 base pairs, at least about 300 base pairs, at least about 400 base pairs, at least about 500 base pairs, at least about 600 base pairs, at least about 700 base pairs, at least about 800 base pairs, at least about 900 base pairs, or at least about 1000 base pairs. In some embodiments, the primers are for an amplicon of about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 base pairs.

[0146] In some embodiments, the amplification reagent comprises a magnesium salt, such as magnesium chloride or magnesium sulfate. In some embodiments, the amplification reagent comprises magnesium chloride.

[0147] In some embodiments, the amplification reagent includes a buffer. Exemplary buffers include tris(hydroxymethyl)aminomethane (Tris) buffer. The buffer may further include a salt, such as an ammonium salt (e.g., ammonium chloride or ammonium sulfate) and / or a potassium salt (e.g., potassium chloride). The pH of the buffer may be specifically adjusted for the DNA polymerase used in the amplification reaction. In some embodiments, the buffer has a pH of about 7.5 to about 10, or about 8.0 to about 9.5.

[0148] In some embodiments, such as those of the method for detecting viable microorganisms in a sample, the amplification reagents include one or more probes, or any nucleic acid conjugated to at least one label, such as at least one dye. The probe can be a sequence-specific binding partner for the nucleic acid target and / or amplicon. The probe can be designed to enable detection of target amplification based on fluorescence resonance energy transfer (FRET), including one or more nucleic acids linked to a pair of dyes that collectively exhibit fluorescence resonance energy transfer (FRET) when brought into close proximity with each other. The pair of dyes can provide, among other things, a first and second emitter, or an emitter and a quencher. Fluorescence emission from the pair of dyes changes when the dyes are separated from each other, such as by cleavage of the probe during primer extension (e.g., a 5' nuclease assay using a TAQMAN probe, etc.), or when the probe hybridizes to an amplicon (e.g., a molecular beacon probe). The nucleic acid portion of the probe can have any suitable structure or origin; for example, it can be a locked nucleic acid, a member of a universal probe library, etc. In other cases, the probe and one of the pair of primers can be combined within the same molecule. For example, a primer-probe molecule may contain a primer sequence at its 3' end and a molecular beacon-type probe at its 5' end. This configuration allows related primer-probe molecules labeled with different dyes to be used in a multiplex assay with the same reverse primer to quantify target sequences that differ by a single nucleotide (single nucleotide polymorphism (SNP)).

[0149] In some embodiments, the amplification reagents also include one or more label or reporter molecules. Exemplary dyes used for labeling are fluorescent dyes (fluorophores) and fluorescence quenchers. Reporters include any compound or set of compounds that report a state, such as the extent of a reaction. Exemplary reporters include at least one dye, such as a fluorescent dye or energy transfer pair, and / or at least one oligonucleotide. Exemplary reporters for nucleic acid amplification assays may include probes and / or intercalating dyes (e.g., SYBR Green, ethidium bromide, etc.).

[0150] According to the embodiments provided herein, the concentrations of the amplification reagents described above can vary depending on the particular reaction conditions and reagents used, as well as the desired target to be amplified. One of ordinary skill in the art will readily recognize that any particular concentration or concentration range provided herein for any amplification reagent, including concentration ranges for compounds of the present disclosure, will vary depending on the particular reaction conditions and reagents used and is not meant to be limiting.

[0151] In some embodiments, such as those involving PCR, after adding the DNA polymerase and amplification reagents to form a mixture, the method further comprises heating the mixture to a temperature of at least 90°C to activate the DNA polymerase before subjecting the mixture to a thermal cycling protocol. For example, the DNA polymerase may be initially unreactive at ambient temperature through inhibition by antibody interactions or other modifications. The first step activates DNA polymerization by causing dissociation from the inhibitor. In this step, the mixture can be subjected to a temperature of at least 90°C, e.g., from about 90°C to about 96°C, e.g., about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, or about 96°C. This heating step can be performed for about 1 minute to about 10 minutes, or from about 2 minutes to about 5 minutes, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

[0152] In some embodiments, the nucleic acid is amplified using a thermal cycling protocol. In some embodiments, the thermal cycling protocol comprises: (1) a denaturation step comprising subjecting the mixture to a temperature of about 90-96°C; (2) an annealing step comprising subjecting the mixture to a temperature of about 45-68°C; and (3) an extension step comprising subjecting the mixture to a temperature of about 50-72°C; The series of steps (1) to (3) is repeated at least 10 times consecutively.

[0153] The denaturation step involves subjecting the mixture to a temperature of about 90-96°C to denature the double-stranded DNA and allow subsequent primer annealing. For example, the mixture can be subjected to a temperature of about 90°C to about 96°C, e.g., about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, or about 96°C. This step can be performed for about 15 seconds to about 60 seconds, e.g., about 15 seconds, about 30 seconds, about 45 seconds, or about 60 seconds.

[0154] The annealing step involves subjecting the mixture to a temperature of about 45°C to 68°C to allow the primers to bind to their complementary sequences on the denatured DNA. The optimal annealing temperature depends on the specific primers used and is typically about 5°C below the melting temperature (Tm) of the primers. For example, the mixture can be subjected to a temperature of about 45°C to about 68°C, e.g., about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, or about 68°C. The annealing step can be performed for about 15 seconds to about 60 seconds, e.g., about 15 seconds, about 30 seconds, about 45 seconds, or about 60 seconds.

[0155] The extension step involves subjecting the mixture to a temperature of about 50-72°C to allow the DNA polymerase to extend the DNA strand starting from the annealed primer. The extension temperature depends on the specific DNA polymerase used, and the extension time depends on the length of the amplicon. Typical extension temperatures are about 50°C to about 72°C, e.g., about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, or about 72°C. A typical extension time is about 1 minute per kilobase of DNA. For example, the extension step can be carried out for about 15 seconds to about 2 minutes, such as about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 75 seconds, about 90 seconds, about 105 seconds, or about 120 seconds.

[0156] The sequence of denaturing, annealing, and extension steps can be repeated about 10 or more times in succession, for example, about 10, 15, 20, 25, 30, 35, 40, 45, or 50 times in succession.

[0157] In other embodiments, the thermal cycling protocol may include only two steps: a denaturation step and a combined annealing / extension step. For example, in some embodiments, the thermal cycling protocol may include: (1a) a denaturation step comprising subjecting the mixture to a temperature of 90-96°C; (2a) an annealing / extension step comprising subjecting the mixture to a temperature of 45-70°C; The series of steps (1a) to (2a) is repeated 10 or more times consecutively.

[0158] In these embodiments, the annealing / extension step comprises subjecting the mixture to a temperature of about 45° C. to about 70° C., e.g., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., or about 70° C. The annealing / extension step can be carried out for about 15 seconds to about 2 minutes, e.g., about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 75 seconds, about 90 seconds, about 105 seconds, or about 120 seconds.

[0159] The sequence of denaturation and annealing / extension steps can be repeated about 10 or more times in succession, for example, about 10, 15, 20, 25, 30, 35, 40, 45, or 50 times in succession.

[0160] In some embodiments, the nucleic acid is amplified using an isothermal amplification protocol.

[0161] In methods for detecting viable microorganisms or cells in a sample, the method generates a detectable signal, the signal indicating the presence of viable organisms or cells in the sample. In some embodiments of such methods, the method further comprises detecting the detectable signal from the sample. The particular detection step will depend on the probes and / or dyes used in the method. Any suitable detection method can be used, such as photochemical, biochemical, immunochemical, electrical, optical, or chemical means. In some embodiments, the detection step is a fluorescence detection step. For example, in some embodiments, the amplification product can be directly detected using fluorescence. In some embodiments, a fluorescent probe of the amplification product can be detected using fluorescence.

[0162] In some embodiments, detection is performed using a spectrophotometric thermal cycler, such as those commercially available from Agilent (e.g., AriaDx and AriaMx instruments), Applied Biosystems (e.g., QuantStudio® systems), Bio-Rad (e.g., CFX systems), Cepheid (e.g., SmartCycler®), Roche (e.g., LightCycler® systems), and Stratagene (e.g., Mx3005p).

[0163] In some embodiments, the present disclosure provides methods for removing nucleic acids from a sample, the methods comprising contacting the sample with a compound described herein (i.e., a compound comprising a NAB moiety, an LDCD moiety, and a NAM moiety). In some embodiments of such methods, the compound is immobilized on a solid support, such as a bead, a resin, or a membrane. Such methods can be used with any sample from which it is desirable to remove free nucleic acids. For example, certain reagents or solutions intended for use as human injectables can be treated with the compounds described herein to remove free DNA.

[0164] 5. Working Example It will be appreciated by those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and recognizable and may be made using appropriate equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. The present disclosure will be more clearly understood by reference to the following examples, which are intended merely to illustrate some aspects and embodiments of the disclosure and should not be construed as limiting the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referenced herein are hereby incorporated by reference in their entirety.

[0165] Example 1 Viability PCR workflow A diagram of the viability PCR (vPCR) workflow described in this application is shown in Figure 1. 1. In this procedure, a live-cell-impermeable nucleic acid-modifying compound is incubated with a mixture of bacterial cells, and due to the permeability of the compromised cell membrane, it enters only dead / dying cells. As a result, only nucleic acids from non-viable cells are covalently crosslinked, preventing subsequent amplification by PCR. Therefore, the signal observed in the vPCR detection assay comes only from viable cells. Dead cells are indicated by perforated gray membranes, while live cells are indicated by intact purple membranes.

[0166] Example 2 compound synthesis Abbreviations used in this example include: Ac is acetyl; ACN is acetonitrile; DIPEA is N,N-diisopropylethylamine; DMF is N,N-dimethylformamide; HATU is (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HPLC is high performance liquid chromatography; RT is room temperature; TFA is trifluoroacetic acid; TIPS is triisopropylsilane; TMS is trimethylsilyl; and TSTU is N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate. Compound CS0729. [ka]

[0167] Step 1: 3,8-Diamino-5-(4-carboxybenzyl)-6-phenylphenanthridine-5-ium bromide (50 mg, 0.1 mmol, 1.0 equiv.) was added to a solution of tert-butyl (2-aminoethyl)carbamate (24 mg, 0.15 mmol, 1.5 equiv.), DIPEA (39 mg, 0.3 mmol, 3.0 equiv.), and HATU (57 mg, 0.15 mmol, 1.5 equiv.) in DMF (2 mL). The solution was stirred at room temperature for 3 h and quenched by the addition of 2 mL of CHCN / 0.1% TFA (1 / 1) in HO. The mixture was then purified by reverse phase HPLC to give 3,8-diamino-5-(4-((2-((tert-butoxycarbonyl)amino)-ethyl)carbamoyl)benzyl)-6-phenylphenanthridin-5-ium 2,2,2-trifluoroacetate.

[0168] Step 2: 3,8-Diamino-5-(4-((2-((tert-butoxycarbonyl)amino)-ethyl)carbamoyl)benzyl)-6-phenylphenanthridin-5-ium 2,2,2-trifluoroacetate (67.5 mg, 0.1 mmol, 1.0 equiv) was dissolved in TFA (1 mL) and TIPS (0.1 mL) and stirred for 30 minutes. The reaction was concentrated in vacuo and purified by reverse-phase HPLC to give 5-(4-((2-(14-azanyl)ethyl)carbamoyl)benzyl)-3,8-diamino-6-phenylphenanthridin-5-ium 2,2,2-trifluoroacetate.

[0169] Step 3: 5-(4-((2-(14-Azanyl)ethyl)carbamoyl)benzyl)-3,8-diamino-6-phenylphenanthridin-5-ium 2,2,2-trifluoroacetate (69 mg, 0.1 mmol, 1.0 equiv.) was added to a solution of 4-(4-(bis(2-chloroethyl)amino)-phenyl)butanoic acid (33 mg, 0.11 mmol, 1.1 equiv.), HATU (42 mg, 0.11 mmol, 1.1 equiv.), and DIPEA (39 mg, 0.3 mmol, 3.0 equiv.) in DMF (2 mL). The solution was stirred at room temperature for 3 h and quenched by addition to 2 mL of CHCN / 0.1% TFA (1 / 1) in HO. The mixture was then purified by reverse-phase HPLC to give 3,8-diamino-5-(4-((2-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanamido)ethyl)carbamoyl)-benzyl)-6-phenylphenanthridin-5-ium 2,2,2-trifluoroacetate (CS0729).

[0170] Compounds CS0727, CS0775, CS0776, CS0777, CS0935, and CS0942 were similarly synthesized using the above procedure, and their structures and characterization data are shown in Table 1. [Table 1-1] [Table 1-2]

[0171] Compound CS0733 [ka]

[0172] Step 1: 5-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)pentanoic acid trihydrobromide (76.6 mg, 0.1 mmol, 1.0 equiv.) was added to a solution of tert-butyl (2-aminoethyl)carbamate (24 mg, 0.15 mmol, 1.5 equiv.), DIPEA (78 mg, 0.6 mmol, 6.0 equiv.), and HATU (57 mg, 0.15 mmol, 1.5 equiv.) in DMF (2 mL). The solution was stirred at room temperature for 3 h and quenched by the addition of 2 mL of CHCN / 0.1% TFA (1 / 1) in HO. The mixture was then purified by silica gel purification to give tert-butyl (2-(5-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)pentanamido)ethyl)carbamate.

[0173] Step 2: tert-Butyl (2-(5-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)-pentanamido)ethyl)carbamate (67 mg, 0.1 mmol, 1.0 equiv) was dissolved in TFA (1 mL) and TIPS (0.1 mL) and stirred for 30 minutes. The reaction was concentrated in vacuo and purified by reverse-phase HPLC to give N-(2-aminoethyl)-5-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)pentanamido tetrakis(2,2,2-trifluoroacetate).

[0174] Step 3: N-(2-aminoethyl)-5-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phen-oxy)pentanamide tetrakis(2,2,2-trifluoroacetate) (101 mg, 0.1 mmol, 1.0 equiv) was added to a solution of 4-(4-(bis(2-chloroethyl)amino)-phenyl)butanoic acid (33 mg, 0.11 mmol, 1.1 equiv), HATU (42 mg, 0.11 mmol, 1.1 equiv), DIPEA (117 mg, 0.9 mmol, 9.0 equiv) in DMF (2 mL). The solution was stirred at room temperature for 3 h and then concentrated in vacuo. The mixture was then purified on normal phase silica gel to give (CS0733).

[0175] Compound CS0717 was similarly synthesized using the procedure described above, and its structure and characterization data are shown in Table 2. [Table 2]

[0176] Compound CS0868 [ka]

[0177] Step 1: A solution of 3-(4-(5-(4-methylpiperazin-1-yl)-1H,3'H-[2,5'-bibenzo[d]imidazol]-2'-yl)phenoxy)propan-1-aminetetrakis(2,2,2-trifluoroacetate) (93.7 mg, 0.1 mmol, 1.0 equiv) in DMF (1 mL) was added to a solution of tert-butyl acrylate (39 mg, 0.3 mmol, 3.0 equiv) and DIPEA (65 mg, 0.5 mmol, 5.0 equiv) in DMF (1 mL). The solution was stirred at room temperature for 48 h. The product was isolated using reverse-phase HPLC to give tert-butyl 3-((3-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)propyl)-amino)propanoate tris(2,2,2-trifluoroacetate).

[0178] Step 2: tert-Butyl 3-((3-(4-(5-(4-methylpiperazin-1-yl))-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)-propyl)-amino)propanoate tris(2,2,2-trifluoroacetate) (95 mg, 0.1 mmol, 1.0 equiv.) was dissolved in TFA (1 mL) and TIPS (0.1 mL) and stirred for 30 minutes. The reaction was concentrated in vacuo and purified by reverse-phase HPLC to give 3-((3-(4-(5-(4-methylpiperazin-1-yl))-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)propyl)amino)propanoic acid (4:1) with the 2,2,2-trifluoroacetate compound.

[0179] Step 3: N-(2-aminoethyl)-5-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phen-oxy)pentanamide tetrakis(2,2,2-trifluoroacetate) (101 mg, 0.1 mmol, 1.0 equiv) was added to a solution of 2-(4-(bis(2-chloroethyl)amino)-phenoxy)acetic acid (33 mg, 0.11 mmol, 1.1 equiv), HATU (42 mg, 0.11 mmol, 1.1 equiv), and DIPEA (117 mg, 0.9 mmol, 9.0 equiv) in DMF (2 mL). The solution was stirred at room temperature for 3 hours and then concentrated in vacuo. The mixture was then purified using reverse-phase preparative HPLC to give 2,2,2-trifluoroacetic acid compound having 3-(2-(4-(bis(2-chloroethyl)amino)phenoxy)-N-(3-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)propyl)acetamido)propanoic acid (3:1) (CS0868). MS(M+) 852.9.

[0180] Compound CS0858 [ka]

[0181] Step 1: tert-Butyl (3-oxopropyl)carbamate (17 mg, 0.1 mmol, 1.0 equiv.) was dissolved in NaBH(OAc) (53 mg, 0.25 mmol, 2.5 equiv.) and AcOH (240 μL, 4.0 mmol, 40 equiv.) in ACN (2 mL) and stirred for 30 min. After that, a solution of 3-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phen-oxy)propan-1-amine (48 mg, 0.1 mmol, 1.0 equiv.) was added. The reaction was stirred at room temperature for 24 hours and purified by reverse-phase preparative HPLC to give the intermediate tert-butyl (3-(3-(bis(benzyloxy)phosphoryl)-N-(3-(4-(5-(4-methylpiperazin-1-yl)-1H,1′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)propyl)propanamido)propyl)carbamate.

[0182] Step 2: 3-(Bis(benzyloxy)phosphoryl)propanoic acid (50 mg, 0.15 mmol, 1.5 equiv) was mixed with TSTU (45 mg, 0.15 mmol, 1.5 equiv) and DIPEA (50 μL, 0.3 mmol, 3.0 equiv) in DMF (2 mL). To this solution was added tert-butyl (3-(3-(bis(benzyloxy)phosphoryl)-N-(3-(4-(5-(4-methyl-piperazin-1-yl)-1H,1′H-[2,5′-bibenzo-[d]imidazol]-2′-yl)phenoxy)propyl)propanamido)propyl)-c-albamate (64 mg, 0.1 mmol, 1.0 equiv.), the solution was stirred at room temperature for 3 hours, and purified by reverse-phase preparative HPLC to provide the intermediate tert-butyl (3-(3-(bis(benzyloxy)phosphoryl)-N-(3-(4-(5-(4-methylpiperazin-1-yl)-1H,1′H-[2,5′-bibenzo[d]imidazo]-2′-yl)phenoxy)propyl)propanamido)propyl)carbamate.

[0183] Step 3: tert-Butyl (3-(3-(bis(benzyloxy)phosphoryl)-N-(3-(4-(5-(4-methylpiperazin-1-yl)-1H,1′H-[2,5′-bibenzo[d]imidazo]-2′-yl)phenoxy)propyl)propanamido)propyl)carbamate (95 mg, 0.1 mmol, 1.0 equiv) was dissolved in TMSBr (2 mL) and stirred at room temperature for 2 h. LC-MS showed complete conversion to the desired product. The reaction was concentrated and purified by reverse phase HPLC purification to give the trifluoroacetate salt of (3-((3-aminopropyl)(3-(4-(5-(4-methylpiperazin-1-yl))-1H,1′H-[2,5′-bibenzo-[d]imidazol]-2′-yl)phenoxy)propyl)amino)-3-oxopropyl)phosphonic acid.

[0184] Step 4: (3-((3-aminopropyl)(3-(4-(5-(4-methylpiperazin-1-yl)-1H,1′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)propyl)amino)-3-oxopropyl)phosphonic acid trifluoroacetate (113 mg, 0.1 mmol, 1.0 equiv) was added to a solution of 2-(4-(bis(2-chloroethyl))amino)phenoxy)acetic acid (33 mg, 0.11 mmol, 1.1 equiv), HATU (42 mg, 0.11 mmol, 1.1 equiv), and DIPEA (117 mg, 0.9 mmol, 9.0 equiv) in DMF (2 mL). The solution was stirred at room temperature for 3 hours and then concentrated in vacuo. The mixture was then purified using reverse-phase preparative HPLC to give the desired trifluoroacetate salt of (3-((3-(2-(4-(bis(2-chloroethyl)amino)phenoxy)acetamido)propyl)(3-(4-(5-(4-methylpiperazin-1-yl)-1H,1′H-[2,5′-bibenzo-[d]imidazol]-2′-yl)phenoxy)propyl)amino)-3-oxopropyl)phosphonic acid (CS0858). MS(M+) 948.3.

[0185] Compound CS0985 [ka]

[0186] Step 1: 3,8-Bis((tert-butoxycarbonyl)amino)-5-(3-iodopropyl)-6-phenylphenanthridine-5-ium iodide (18 mg, 23 μmol, 1.0 equiv.) was mixed with di-tert-butyl((methylazanediyl)bis(ethane-2,1-diyl))dicarbamate (73 mg, 230 μmol, 10 equiv.) in CHCN (2 mL) at 40° C. for 6 days. The reaction was concentrated and purified by reverse preparative HPLC to give 5-(3-(bis(2-((tert-butoxycarbonyl)amino)ethyl)(methyl)ammonio)propyl)-3,8-bis((tert-butoxycarbonyl))amino)-6-phenylphenanthridine-5-ium iodide.

[0187] Step 2: 5-(3-(bis(2-((tert-butoxycarbonyl)amino)ethyl)(methyl)ammonio)propyl)-3,8-bis((tert-butoxycarbonyl)amino)-6-phenylphenanthridin-5-ium iodide (25 mg, 23 μmol, 1.0 equiv.) was dissolved in CFCOH / TIPS (2 mL / 0.2 mL) and stirred at room temperature for 1 h. The reaction was concentrated and purified by reverse preparative HPLC to give the trifluoroacetate salt of 3,8-diamino-5-(3-(bis(2-aminoethyl)-(methyl)ammonio)propyl)-6-phenylphenanthridin-5-ium.

[0188] Step 3: 3,8-Diamino-5-(3-(bis(2-aminoethyl)-(methyl)ammonio)propyl)-6-phenylphenanthridin-5-ium trifluoroacetate (44 mg, 0.1 mmol, 1.0 equiv) was added to a solution of 4-(4-(bis(2-chloroethyl)amino)phenoxy)butanoic acid (76 mg, 0.24 mmol, 2.4 equiv), HATU (91 mg, 0.24 mmol, 2.4 equiv) and DIPEA (117 mg, 0.9 mmol, 9.0 equiv) in DMF (2 mL). The solution was stirred at room temperature for 3 hours and then purified using reverse-phase preparative HPLC to give the desired trifluoroacetate salt of 3,8-diamino-5-(3-(bis(2-(4-(4-(bis(2-chloroethyl)amino)phenoxy)butanamido)ethyl)(methyl)ammonio)propyl)-6-phenylphenanthridin-5-ium (CS0985). MS(M+)=524.3.

[0189] Compound CS1065 [ka]

[0190] Step 1: (E)-2-((1-benzyl-2-(bis(3-(dimethylamino)propyl)amino)quinolin-4(1H)-ylidene)methyl)-5,6-dihydro-4H-thiazolo[5,4,3-ij]quinolin-3-ium trifluoroacetate (70 mg, 0.1 mmol, 1.0 equiv) was added to a solution of tert-butyl bromoacetate (390 mg, 2 mmol, 20 equiv) in CHCN (5 mL) and the reaction was heated at 50° C. for 3 days. The reaction was then concentrated and used directly in the next step without further purification.

[0191] The above intermediate (E)-2-((1-benzyl-2-(bis(3-((2-(tert-butoxy)-2-oxoethyl)dimethylammonio)propyl)amino)quinolin-4(1H))-ylidene)methyl)-5,6-dihydro-4H-thiazolo[5,4,3-ij]quinolin-3-ium trifluoroacetate (11.6 mg, 0.01 mmol, 1.0 equiv.) was dissolved in TFA / TIPS (2 / 0.2 mL) and stirred at room temperature for 2 hours. The reaction was concentrated and purified by reverse-phase preparative HPLC to give (E)-2-((1-benzyl-2-(bis(3-((carboxymethyl)dimethylammonio)propyl)amino)quinolin-4(1H)-ylidene)methyl)-5,6-dihydro-4H-thiazolo[5,4,3-ij]quinolin-3-ium trifluoroacetate.

[0192] Step 2: 2-(4-(bis(2-chloroethyl)amino)phenoxy)acetic acid (29 mg, 0.1 mmol, 1.0 equiv) was added to a solution of tert-butyl(2-aminoethyl)carbamate (160 mg, 0.1 mmol, 1.0 equiv), TSTU (33 mg, 0.11 mmol, 1.1 equiv), and DIPEA (26 mg, 0.2 mmol, 2.0 equiv) in CHCN (2 mL). The reaction was concentrated in vacuo and purified on normal phase silica gel (heptane / EtOAc) to give the intermediate tert-butyl(2-(2-(4-(bis(2-chloroethyl)amino)phenoxy)acetamido)ethyl)carbamate, which was subsequently dissolved in TFA / TIPS (2 / 0.2 mL). The reaction was stirred for 30 minutes to ensure completion and concentrated in vacuo to give N-(2-aminoethyl)-2-(4-(bis(2-chloro-ethyl)amino)phenoxy)acetamide, trifluoroacetate salt product, which was used in the next step without further purification.

[0193] Step 3: N-(2-aminoethyl)-2-(4-(bis(2-chloro-ethyl)amino)phenoxy)acetamide, trifluoroacetate (8.8 mg, 0.02 mmol, 2.0 equiv.) was added to a solution of (E)-2-((1-benzyl-2-(bis(3-((carboxymethyl)dimethylammonio)propyl)amino)quinolin-4(1H)-ylidene)methyl)-5,6-dihydro-4H-thiazolo[5,4,3-ij]quinolin-3-ium trifluoroacetate (10.5 mg, 0.1 mmol, 1.0 equiv.), HATU (84 mg, 0.22 mmol, 2.2 equiv.), and DIPEA (240 mg, 1.8 mmol, 18.0 equiv.) in DMF (2 mL). The solution was stirred at room temperature for 3 hours and then concentrated in vacuo. The mixture was then purified using reverse-phase preparative HPLC to give (E)-2-((1-benzyl-2-(bis(3-((2-((2-(2-(4-(bis(2-chloroethyl)amino)phenoxy)acetamido)ethyl)amino)-2-oxoethyl)dimethylammoni-o)propyl)amino)quinolin-4(1H)-ylidene)methyl)-5,6-dihydro-4H-thiazolo[5,4,3-ij]quinolin-3-ium 2,2,2-trifluoroacetate (CS1065). MS(M+)=447.5.

[0194] Example 3 Listeria innocua viability PCR experiment A turbid overnight culture of Listeria innocua was subcultured into Terrific Broth, grown to early / mid-exponential phase, and concentrated to an OD of approximately 1.0 (approximately 1 x 10^8 CFU / mL). 200 μL of live or dead Listeria innocua cells (heat-killed at 95°C for 15 minutes, HK) was incubated with the indicated concentrations of compound CS0775 at 37°C for 90 minutes. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and dye-based qPCR analysis was performed using primers specific for Listeria innocua. Results are shown in Figure 2. ΔCt is defined as the difference in qPCR signal threshold between dead and live cells. Live and non-viable cells were clearly distinguished using the described viability PCR workflow.

[0195] Example 4 Effect of solvents on permeation of non-viable cells A turbid culture of Pseudomonas aeruginosa was subcultured into LB broth, grown to early / mid-exponential phase, and concentrated to an OD of approximately 1.0 (approximately 1 x 10^8 CFU / mL). 200 μL of live or dead Pseudomonas aeruginosa cells (heat-killed at 95°C for 15 minutes, HK) was incubated with 20 μM CS0775 at 37°C for 90 minutes. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and dye-based qPCR analysis was performed using a primer set specific for Pseudomonas aeruginosa. The results are shown in Figure 3. PCR amplicon size is plotted on the horizontal axis. ΔCt is defined as the difference in qPCR signal threshold between dead and live cells. Increasing the DMSO concentration in the final reaction increases DNA modification efficiency and subsequently improves the discrimination of non-viable from viable cells.

[0196] Example 5 Amplicon length analysis Turbid cultures of Pseudomonas aeruginosa and Listeria innocua were subcultured into LB Broth or Terrific Broth, respectively, grown to early / mid-exponential phase, and concentrated to an OD of approximately 1.0 (approximately 1 x 10^8 CFU / mL). 200 μL of live or dead bacterial cells (heat-killed at 95°C for 15 minutes, HK) were incubated with 10 μM (Listeria) or 50 μM (Pseudomonas) CS0775 for 90 minutes at 37°C. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and dye-based qPCR analysis was performed using a primer set specific for Listeria innocua. Results are shown in Figure 4. PCR amplicon size is plotted on the horizontal axis. The live / dead discrimination (ΔCt) increases with amplicon length.

[0197] Example 6 Effect of compound concentration Turbid cultures of Pseudomonas aeruginosa, Escherichia coli, Legionella pneumophila, and Listeria innocua were subcultured into LB broth (E. coli, P. aeruginosa), terrific broth (L. innocua), or Legionella broth (L. pneumophila) and concentrated to early / mid-exponential phase and an OD of approximately 1.0 (approximately 1 x 10^8 CFU / mL). 200 μL of live or dead bacterial cells (heat-killed at 95°C for 15 minutes, HK) were incubated with the indicated concentrations of CS0775 at 37°C for 90 minutes. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and dye-based qPCR analysis was performed using primer sets specific for the indicated bacterial species. Results are shown in Figure 5. The optimal concentrations of compounds differ between Gram-negative and Gram-positive bacteria.

[0198] Example 7 Viability PCR assay using various compounds Turbid cultures of Pseudomonas aeruginosa, Legionella pneumophila, and Listeria innocua were subcultured into LB broth (P. aeruginosa), terrific broth (L. innocua), or Legionella broth (L. pneumophila) and concentrated to early / mid-exponential phase and an OD of approximately 1.0 (approximately 1 x 10^8 CFU / mL). 200 μL of live or dead bacterial cells (heat-killed at 95°C for 15 minutes, HK) at the indicated concentrations were incubated at 37°C for 90 minutes. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and dye-based qPCR analysis was performed using primer sets specific for the indicated bacterial species. Results are shown in Figure 6. Different classes of compounds can effectively distinguish between live and non-viable cells when used as part of a viability PCR procedure.

[0199] Example 8 Assay Sensitivity Turbid cultures of Legionella pneumophila were subcultured into Legionella Broth, grown to early / mid-exponential phase, and concentrated to an OD of approximately 1.0 (approximately 1x10^8 CFU / mL). Serial dilutions were performed, and 200uL of the indicated concentrations of viable Legionella pneumophila cells were incubated with 10uM CS0775 at 37°C for 90 minutes. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and probe-based qPCR analysis was performed using primer and probe sets specific for Legionella pneumophila. Results are shown in Figure 7. This assay yielded approximately 10 2 It can detect bacteria at concentrations as low as CFU / mL.

[0200] Example 9 Assays for samples containing live and dead cells Turbid cultures of Legionella pneumophila were subcultured into Legionella Broth, grown to early / mid-exponential phase, and concentrated to an OD of approximately 1.0 (approximately 1 x 10^8 CFU / mL). Cells were then diluted to 10^5 CFU / mL. Live or dead bacterial cells (heat-killed at 95°C for 15 minutes, HK) at the indicated ratios were mixed (total volume: 200 μL) and incubated with 10 μM CS0775 at 37°C for 90 minutes. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and probe-based qPCR analysis was performed using a primer and probe set specific for Legionella pneumophila. The results are shown in Figure 8. This assay can distinguish and detect a small percentage of live cells in a background of primarily dead cells.

[0201] Example 10 Comparison of viability PCR with conventional culture Turbid cultures of Legionella pneumophila were subcultured into Legionella Broth, grown to early / mid-exponential phase, and concentrated to an OD of approximately 1.0 (approximately 1 x 10^8 CFU / mL). Serial dilutions were performed and plated on Legionella agar to confirm CFU / mL. In parallel, 200 μL of the indicated dilutions of live Legionella pneumophila cells were incubated at 37°C for 90 minutes with or without 10 μM CS0775. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and probe-based qPCR analysis was performed using a primer and probe set specific for Legionella pneumophila. A standard curve of Legionella template DNA was amplified simultaneously with the experimental samples to allow for quantification. Results are shown in Table 2. vGU: raw genome unit. This assay generates quantitative data comparable to traditional culture-based Legionella pneumophila detection techniques. [Table 3]

[0202] Example 11 Viability PCR experiments using adeno-associated virus (AAV). AAV9 reference capsids containing a recombinant CMV-GFP plasmid (Vigene Biosciences; Rockville, MD) were diluted to approximately 10^8 / mL in PBS. 200 μL of live (intact) or dead (heat-inactivated at 75°C for 10 minutes, HI) AAV9 suspension was incubated with 1 μM CS0775 at 37°C for 60 minutes. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 minutes. Nucleic acids were purified using the Maxwell automated purification workflow, and dye-based qPCR analysis using primers specific for GFP was performed. The results are shown in Figure 9. Intact and heat-inactivated viral capsids were clearly distinguished using the described viability PCR workflow.

[0203] It should be understood that the above detailed description and accompanying examples are illustrative only and should not be construed as limitations on the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0204] It will be apparent to those skilled in the art that various changes and modifications can be made to the disclosed embodiments. Such changes and modifications, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the present disclosure, can be made without departing from the spirit and scope thereof.

Claims

1. A compound or a salt thereof, wherein the compound is (A) Nucleic acid binding portion (“NAB portion”), (B) a live / dead cell discrimination moiety ("LDCD moiety"); (C) The compound or a salt thereof, comprising a nucleic acid modifying moiety ("NAM moiety").

2. The compound has the following structure: A-B-C wherein A is the NAB moiety, B is the LDCD moiety, and C is the nucleic acid modifying moiety.

2. The compound of claim 1, or a salt thereof, having the formula:

3. 2. The compound or salt thereof according to claim 1, wherein the compound has two or more NAB moieties, two or more LDCD moieties, and / or two or more NAM moieties.

4. 4. The compound or salt thereof according to any one of claims 1 to 3, wherein the NAB moiety is a groove-binding moiety, an intercalating moiety, or a mixed-mode binding moiety.

5. The compound or salt thereof according to any one of claims 1 to 4, wherein the NAB moiety comprises a bibenzimidazole moiety or a phenylphenanthridium moiety.

6. The NAB portion is 【Chemistry 1】 The compound according to any one of claims 1 to 4, or a salt thereof, having a structure selected from:

7. 7. The compound or salt thereof of any one of claims 1 to 6, wherein the NAM moiety comprises a bischloroethylamine (nitrogen mustard) moiety, a platinum-based moiety, a 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indolyl moiety, or a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) moiety.

8. The NAM portion is 【Chemistry 2】 The compound according to any one of claims 1 to 7, or a salt thereof, having a structure selected from:

9. 9. The compound or salt thereof according to any one of claims 1 to 8, wherein the LDCD moiety comprises at least one charged moiety.

10. The compound of claim 9 , wherein the LDCD moiety comprises at least one quaternary ammonium group.

11. The compound or salt thereof according to any one of claims 1 to 8, wherein the LDCD moiety comprises at least one poly(ethylene glycol) moiety.

12. The poly(ethylene glycol) moiety has the formula: -(CH) 2 CH 2 O) n - wherein n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

12. The compound of claim 11 having the formula:

13. 9. The compound or salt thereof according to any one of claims 1 to 8, wherein the LDCD moiety comprises a functional group attached to a solid support.

14. 9. The compound or salt thereof according to any one of claims 1 to 8, wherein the LDCD moiety comprises a metabolically cleavable group.

15. The compound is 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 [Chemistry 3-4] The compound according to any one of claims 1 to 8, selected from:

16. 1. A method for detecting viable microorganisms or cells in a sample, comprising: (a) contacting the sample with a compound according to any one of claims 1 to 15 or a salt thereof to form a first mixture; (b) contacting the first mixture with an inactivating agent to form a second mixture; (c) amplifying nucleic acids from the second mixture to produce a detectable signal; wherein the signal indicates the presence of viable microorganisms or living cells in the sample.

17. 17. The method of claim 16, which does not include a photoactivation step.

18. The method according to claim 16 or 17, which does not include a culture step.

19. The method of any one of claims 16 to 18, wherein step (a) comprises contacting the sample with the compound or salt thereof for 5 minutes to 180 minutes.

20. 20. The method of claim 19, wherein step (a) comprises contacting the sample with the compound or salt thereof for 60 to 120 minutes.

21. The method of any one of claims 16 to 20, wherein step (a) comprises adding to the sample a composition comprising the compound or a salt thereof in a solvent.

22. 22. The method of any one of claims 16 to 21, wherein step (a) further comprises contacting the sample with one or more additional compounds according to any one of claims 1 to 15.

23. 23. The method of claim 22, wherein the solvent is dimethyl sulfoxide.

24. 24. The method of any one of claims 16 to 23, wherein in step (a), the first mixture comprises the compound at a concentration of 5 to 100 micromolar.

25. 25. The method of any one of claims 16 to 24, wherein the inactivating agent comprises a nucleophile selected from an amine and a thiol.

26. 26. The method of claim 25, wherein the inactivating agent is selected from cysteine, glutathione, dNTPs, guanine, amine-containing buffers, and any mixture thereof.

27. Step (c) is (i) lysing cells in the second mixture to form a lysed sample; (ii) adding a DNA polymerase and amplification reagents to the lysed sample to form a mixture; (iii) subjecting the mixture to a thermal cycling protocol to amplify the nucleic acid from the sample; The method according to any one of claims 16 to 26, comprising:

28. 28. The method of claim 27, further comprising removing contaminants and / or cellular debris from the lysed sample prior to adding the DNA polymerase and amplification reagents.

29. 29. The method of claim 27 or 28, further comprising heating the mixture to a temperature of at least 90°C to activate the DNA polymerase prior to subjecting the mixture to the thermal cycling protocol.

30. 30. The method of claim 29, wherein the DNA polymerase is a thermostable DNA polymerase selected from Taq, Tbr, Tfi, Tfl, KOD, Tru, Tth, Tli, Tac, Tne, Tma, Pfu, Pho, Pwo, ES4, Bca, Bst, Sac, Sso, Poc, Pab, and Mth, or a mutant, variant, or derivative of any of them.

31. 31. The method of claim 30, wherein the DNA polymerase is Taq polymerase.

32. The method of any one of claims 37 to 31, wherein the amplification reagents comprise at least one primer, deoxynucleotide triphosphates, a buffer, and a magnesium salt.

33. 33. The method of claim 32, wherein the magnesium salt is magnesium chloride.

34. The method of any one of claims 27 to 33, wherein the amplification reagents comprise forward and reverse primers for target amplicons in the sample.

35. The thermal cycling protocol comprises: (1) a denaturation step comprising subjecting the mixture to a temperature of 90-96°C; (2) an annealing step comprising subjecting the mixture to a temperature of 45 to 68°C; (3) an extension step comprising subjecting the mixture to a temperature of 50 to 72°C; The method according to any one of claims 27 to 34, wherein the series of steps (1) to (3) are repeated 10 or more times consecutively.

36. The method according to claim 35, wherein the series of steps (1) to (3) is repeated 20 or more times consecutively.

37. The thermal cycling protocol comprises: (1a) a denaturing step comprising subjecting the mixture to a temperature of 90-96°C; (2a) an annealing / extension step comprising subjecting the mixture to a temperature of 45-70°C; The method according to any one of claims 27 to 34, wherein the series of steps (1a) to (2a) is repeated 10 or more times consecutively.

38. The method according to claim 37, wherein the series of steps (1a) to (2a) is repeated 20 or more times consecutively.

39. Step (c) is (i) lysing cells in the second mixture to form a lysed sample; (ii) adding a DNA polymerase and amplification reagents to the lysed sample to form a mixture; (iii) subjecting the mixture to an isothermal amplification protocol to amplify the nucleic acids from the sample; The method according to any one of claims 16 to 26, comprising:

40. The method of any one of claims 16 to 39, wherein the detectable signal is a fluorescent signal.

41. The method of any one of claims 16 to 40, wherein the microorganism is a bacterium.

42. The bacteria may be Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, En Terobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionell 42. The method of claim 41, wherein the bacterium is from a genus selected from the group consisting of: Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Salmonella, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia.

43. The method of any one of claims 16 to 40, wherein the microorganism is a virus.

44. The virus may be Retroviridae, Picornaviridae, Calciviridae, Flaviridae, Coronaviridae, R habdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arena 44. The method of claim 43, wherein the active ingredient is from a family selected from the families Viridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae.

45. 41. The method of any one of claims 16 to 40, wherein the microorganism or cell is a fungus, yeast, mammalian cell, or plant cell.

46. 1. A method for amplifying nucleic acids from a sample, comprising: (a) contacting the sample with a compound according to any one of claims 1 to 15 or a salt thereof to form a first mixture; (b) contacting the first mixture with an inactivating agent to form a second mixture; (c) amplifying nucleic acids from the second mixture; A method comprising:

47. 47. The method of claim 46, which does not include a photoactivation step.

48. 48. The method of claim 46 or claim 47, which does not include a culture step.

49. 49. The method of any one of claims 46 to 48, wherein step (a) comprises contacting the sample with the compound or salt thereof for between 5 minutes and 180 minutes.

50. 50. The method of claim 49, wherein step (a) comprises contacting the sample with the compound or salt thereof for 60 to 120 minutes.

51. 51. The method of any one of claims 46 to 50, wherein step (a) comprises adding to the sample a composition comprising the compound or a salt thereof in a solvent.

52. 52. The method of claim 51 , wherein the solvent is dimethyl sulfoxide.

53. 53. The method of any one of claims 46 to 52, wherein in step (a), the first mixture comprises the compound at a concentration of 5 to 100 micromolar.

54. 54. The method of any one of claims 46 to 53, wherein the inactivating agent comprises a nucleophile selected from an amine and a thiol.

55. 55. The method of claim 54, wherein the inactivating agent is selected from cysteine, glutathione, dNTPs, guanine, an amine-containing buffer, or any mixture thereof.

56. Step (c) is (i) lysing cells in the second mixture to form a lysed sample; (ii) adding a DNA polymerase and amplification reagents to the lysed sample to form a mixture; (iii) subjecting the mixture to a thermal cycling protocol to amplify the nucleic acid from the sample; 56. The method of any one of claims 46 to 55, comprising:

57. 57. The method of claim 56, further comprising removing contaminants and / or cellular debris from the lysed sample prior to adding the DNA polymerase and amplification reagents.

58. 58. The method of claim 56 or 57, further comprising heating the mixture to a temperature of at least 90°C to activate the DNA polymerase prior to subjecting the mixture to the thermal cycling protocol.

59. 59. The method of any one of claims 56 to 58, wherein the DNA polymerase is a thermostable DNA polymerase selected from Taq, Tbr, Tfi, Tfl, KOD, Tru, Tth, Tli, Tac, Tne, Tma, Pfu, Pho, Pwo, ES4, Bca, Bst, Sac, Sso, Poc, Pab, and Mth, or a mutant, variant, or derivative of any of them.

60. 60. The method of claim 59, wherein the DNA polymerase is Taq polymerase.

61. 61. The method of any one of claims 56 to 60, wherein the amplification reagents comprise at least one primer, deoxynucleotide triphosphates, a buffer, and a magnesium salt.

62. 62. The method of claim 61, wherein the magnesium salt is magnesium chloride.

63. 63. The method of any one of claims 56 to 62, wherein the amplification reagents comprise forward and reverse primers for target amplicons in the sample.

64. The thermal cycling protocol comprises: (1) a denaturation step comprising subjecting the mixture to a temperature of 90-96°C; (2) an annealing step comprising subjecting the mixture to a temperature of 45 to 68°C; (3) an extension step comprising subjecting the mixture to a temperature of 50 to 72°C; The method according to any one of claims 56 to 63, wherein the series of steps (1) to (3) are repeated 10 or more times consecutively.

65. The method according to claim 64, wherein the series of steps (1) to (3) is repeated 20 or more times consecutively.

66. The thermal cycling protocol comprises: (1a) a denaturing step comprising subjecting the mixture to a temperature of 90-96°C; (2a) an annealing / extension step comprising subjecting the mixture to a temperature of 45-70°C; The method according to any one of claims 56 to 63, wherein the series of steps (1a) to (2a) is repeated 10 or more times consecutively.

67. The method according to claim 66, wherein the series of steps (1a) to (2a) is repeated 20 or more times consecutively.

68. Step (c) is (i) lysing cells in the second mixture to form a lysed sample; (ii) adding a DNA polymerase and amplification reagents to the lysed sample to form a mixture; and (iii) subjecting the mixture to an isothermal amplification protocol to amplify the nucleic acids from the sample; 56. The method of any one of claims 46 to 55, comprising:

69. 69. The method of any one of claims 46 to 68, wherein the sample is a sample suspected of containing an organism or cell selected from a bacterium, a virus, a yeast, a fungus, a mammalian cell, or a plant cell.

70. 70. The method of claim 69, wherein the sample is suspected of containing bacteria.

71. The bacteria may be Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, En Terobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionell 71. The method of claim 70, wherein the bacterium is from a genus selected from the group consisting of: Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Salmonella, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia.

72. 70. The method of claim 69, wherein the sample is suspected of containing a virus.

73. The virus may be Retroviridae, Picornaviridae, Calciviridae, Flaviridae, Coronaviridae, R habdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arena 73. The method of claim 72, wherein the active ingredient is from a family selected from the families Viridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae.

74. 1. A method for removing nucleic acids from a sample, comprising: A method comprising the step of contacting said sample with a compound according to any one of claims 1 to 15.

75. 75. The method of claim 74, wherein the compound is immobilized on a solid support.

76. A system or kit comprising the compound or salt thereof according to any one of claims 1 to 15.

77. 77. The system or kit of claim 76, further comprising a DNA polymerase.

78. 78. The system or kit of claim 77, wherein the DNA polymerase is a thermostable DNA polymerase selected from Taq, Tbr, Tfi, Tfl, KOD, Tru, Tth, Tli, Tac, Tne, Tma, Pfu, Pho, Pwo, ES4, Bca, Bst, Sac, Sso, Poc, Pab, and Mth, or a mutant, variant, or derivative of any thereof.

79. 79. The system or kit of claim 78, wherein the DNA polymerase is Taq polymerase.

80. 80. The system or kit of any one of claims 76 to 79, further comprising one or more amplification reagents.

81. 81. The system or kit of claim 80, wherein the one or more amplification reagents are selected from at least one primer, deoxynucleotide triphosphates, a buffer, and a magnesium salt.

82. 82. The system or kit of claim 81, further comprising forward and reverse primers for a target nucleic acid sequence of an organism or cell selected from a bacterium, a virus, a yeast, a fungus, a mammalian cell, or a plant cell.

83. The organism may be Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, Ent. erobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella 83. The system or kit of claim 82, wherein the bacterium is a bacterium from a genus selected from the group consisting of Bacillus, Bacillus subtilis, Bacillus spp. ...

84. The organism is Retroviridae, Picornaviridae, Calciviridae, Flaviridae, Coronaviridae, Rhabdov. iridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, R 83. The system or kit of claim 82, wherein the virus is from a family selected from Eoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae.

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