Nucleic acid cross-linking reagents and uses thereof

Abstract

Provided herein are multifunctional compounds that bind and modify nucleic acids by forming crosslinks between nucleic acid strands. In certain embodiments, the multifunctional nucleic acid binding / modifying compounds are selective for the nucleic acids of non-living cells and viruses (i.e., they are excluded from or degraded within living cells and viruses) and therefore find use in methods for detecting or distinguishing living cells / viruses and nucleic acids therefrom.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,414, filed March 1, 2022, which is incorporated herein by reference.

[0002] Provided herein are multifunctional compounds that bind and / or modify nucleic acids by forming crosslinks between nucleic acid strands. In certain embodiments, the multifunctional nucleic acid binding / modifying compounds are selective for the nucleic acids of non-living cells and viruses (i.e., they are excluded from or degraded within living cells and viruses) and therefore find use in methods for detecting or distinguishing living cells / viruses and nucleic acids therefrom. [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, standard DNA-targeted methods, such as PCR or qPCR and isothermal amplification methods, cannot easily assess cell viability.

[0004] Propidium monoazide (PMA) has been shown to distinguish DNA associated with live cells from that associated with non-live cells in a technique known as viability PCR (vPCR). In samples where both live and dead cells are present, cell membranes are compromised, allowing PMA to access DNA from dead cells. PMA contains a photoreactive azide moiety that upon photolysis covalently modifies DNA, rendering it unavailable as a template for amplification reactions. Lysing any remaining live cells after the photoactivation step exposes the DNA, allowing amplification. However, variations in light spectrum and intensity can lead to sample-to-sample variability, especially in complex and turbid samples. These difficulties limit the widespread use and adoption of vPCR technology. Summary of the Invention

[0005] Provided herein are multifunctional compounds that bind and modify nucleic acids by forming crosslinks between nucleic acid strands. In certain embodiments, the multifunctional nucleic acid binding / modifying compounds are selective for the nucleic acids of non-living cells and viruses (i.e., they are excluded from or degraded within living cells and viruses) and therefore find use in methods for detecting or distinguishing living cells / viruses and nucleic acids therefrom.

[0006] In some embodiments, provided herein is a multifunctional compound or salt thereof, the multifunctional compound comprising: (A) an RNA binding moiety ("RAB moiety"); (B) a live cell / dead cell differentiation moiety ("LDCD moiety"); and (C) a nucleic acid modifying moiety ("NAM moiety"). In some embodiments, the multifunctional compound has the structure: A B C, where A is a RAB moiety, B is a LDCD moiety, and C is a nucleic acid modifying moiety. In some embodiments, a multifunctional compound has one or more RAB moieties, one or more LDCD moieties, and / or one or more NAM moieties.

[0007] In some embodiments, the RAB moiety has formula (I): [ka] or a tautomer or a salt thereof. (In the formula, R 1 and R 2 are each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, and aryl-C1-C6 alkyl, each of which is optionally substituted with 1 to 3 substituents, or R 1 and R 2together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl or heteroaryl ring; and R 3 is absent or is C1-C6 alkyl.

[0008] In some embodiments, R 1 and R 2 are each independently selected from C1-C6 alkyl, and R 3 is absent or is C1-C6 alkyl.

[0009] In some embodiments, R 1 , R 2 , and R 3 are each independently selected from C1-C6 alkyl.

[0010] In some embodiments, R 1 and R 2 are each independently selected from C1-C6 alkyl (e.g., methyl or ethyl), and R 3 does not exist.

[0011] In some embodiments, R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted, monocyclic 5- or 6-membered heterocyclyl or heteroaryl ring having 1, 2, or 3 heteroatoms independently selected from N, O, and S.

[0012] In some embodiments, R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form a pyrrolidinyl, piperidinyl, morpholino, piperazinyl, or imidazolyl ring, each of which is optionally substituted with one substituent (e.g., C1-C6 alkyl, such as methyl).

[0013] In some embodiments, the group -NR in the moiety of formula (I) 1 R 2 R 3 has a formula selected from the following:

[0014] [ka]

[0015] In some embodiments, the RAB portion has a structure selected from the following: [ka]

[0016] 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. In some embodiments, the NAM moiety has a structure selected from the following:

[0017] [ka]

[0018] In some embodiments, the LDCD portion comprises at least one charged moiety. In some embodiments, the LDCD portion comprises at least one quaternary ammonium group. In some embodiments, the LDCD portion comprises at least one poly(ethylene glycol) moiety. In some embodiments, the poly(ethylene glycol) moiety has the formula -(CH2CH2O) n -, where 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.

[0019] In some embodiments, the multifunctional compound is selected from the following and any salts thereof: [ka]

[0020] In some embodiments, provided herein is a method for detecting viable microorganisms or cells in a sample, the method comprising: (a) contacting the sample with a compound or salt thereof according to any one of claims 1-20 to form a first mixture; (b) contacting the first mixture with an inactivating agent to form a second mixture; and (c) amplifying nucleic acid from the second mixture to generate a detectable signal, wherein the signal indicates the presence of viable microorganisms or cells in the sample. In some embodiments, the method does not include a photoactivation step. In some embodiments, the method does not include an incubation step. In some embodiments, step (a) comprises contacting the sample with a multifunctional compound or salt thereof for between 5 minutes and 180 minutes. In some embodiments, step (a) comprises contacting the sample with a multifunctional compound or salt thereof for between 60 minutes and 120 minutes. In some embodiments, step (a) comprises adding a composition comprising a multifunctional compound or salt thereof in a solvent to the sample. In some embodiments, the solvent is dimethylsulfoxide. In some embodiments, in step (a), the first mixture comprises a multifunctional compound at a concentration of 5 to 100 micromolar. In some embodiments, the inactivation agent comprises a nucleophile selected from an amine and a thiol. In some embodiments, the inactivation agent is selected from cysteine, glutathione, dNTPs, guanine, an amine-containing buffer, and any mixture thereof. In some embodiments, step (c) comprises (i) lysing the cells in the second mixture to form a lysed sample; (ii) adding a reverse transcriptase, a DNA replicating enzyme, 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. In some embodiments, the method further comprises removing contaminants and / or cellular debris from the lysed sample prior to adding the reverse transcriptase, the DNA replicating enzyme, and the amplification reagent. In some embodiments, the amplification reagent comprises at least one primer, a deoxynucleotide triphosphate, a buffer, and a magnesium salt.In some embodiments, the amplification reagent comprises a forward primer and a reverse primer for a target amplicon in the sample. In some embodiments, the detectable signal is a fluorescent signal. In some embodiments, the microorganism is an RNA virus. In some embodiments, the RNA virus is of the order Nidovirales, Picornavirales, or Tymovirales. In some embodiments, the RNA virus is of the order Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Caliciviridae, Flaviviridae, or Togaviridae. In some embodiments, the RNA virus is a human pathogen. In some embodiments, the RNA virus is selected from SARS-CoV-2, HIV-1 rhinovirus, hepatitis A virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, and Zika virus.

[0021] In some embodiments, provided herein is a method of removing RNA from a sample, the method comprising contacting the sample with a multifunctional compound comprising a RAB as described herein. In some embodiments, the multifunctional compound is immobilized on a solid support.

[0022] In some embodiments, provided herein is a system or kit comprising a multifunctional compound comprising a RAB as described herein. In some embodiments, the system or kit further comprises a reverse transcriptase and a DNA replicating enzyme. In some embodiments, the system or kit further comprises one or more amplification reagents. In some embodiments, the system or kit further comprises a forward primer and a reverse primer for a target nucleic acid sequence of an RNA virus. In some embodiments, the RNA virus is of the order Nidovirales, Picornavirales, or Tymovirales. In some embodiments, the RNA virus is of the order Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Caliciviridae, Flaviviridae, or Togaviridae. In some embodiments, the RNA virus is a human pathogen. In some embodiments, the RNA virus is selected from SARS-CoV-2, HIV-1 rhinovirus, hepatitis A virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, and Zika virus.

[0023] In some embodiments, provided herein are multifunctional compounds or salts thereof, the multifunctional compounds comprising: (A) a nucleic acid binding moiety ("NAB moiety"); (B) a live cell / dead cell differentiation moiety ("LDCD moiety"); (C) a nucleic acid modifying moiety ("NAM moiety"); and (D) an affinity moiety or conjugation moiety. In some embodiments, the multifunctional compounds have the structure: AB(D)-C, where A is a NAB moiety, B is a LDCD moiety, C is a nucleic acid modifying moiety, and D is an affinity moiety or conjugation moiety. In some embodiments, the multifunctional compounds have one or more NAB moieties, one or more LDCD moieties, and / or one or more NAM moieties. In some embodiments, the NAB is an RNA binding moiety ("RAB moiety").

[0024] In some embodiments, the RAB moiety has the structure of formula (I): [ka] or a tautomer or salt thereof, 1 and R 2 are each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, and aryl-C1-C6 alkyl, each of which is optionally substituted with 1 to 3 substituents, or R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl or heteroaryl ring, and R 3 is absent or is C1-C6 alkyl. In some embodiments, R 1 and R 2 are each independently selected from C1-C6 alkyl, and R 3 is absent or is C1-C6 alkyl. In some embodiments, R 1 , R 2 , and R 3 are each independently selected from C1-C6 alkyl. 1 and R 2are each independently selected from C1-C6 alkyl (e.g., methyl or ethyl), and R 3 is absent. In some embodiments, R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted, monocyclic 5- or 6-membered heterocyclyl or heteroaryl ring having 1, 2, or 3 heteroatoms independently selected from N, O, and S. In some embodiments, R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form a pyrrolidinyl, piperidinyl, morpholino, piperazinyl, or imidazolyl ring, each of which is optionally substituted with one substituent (e.g., C1-C6 alkyl, such as methyl). In some embodiments, the group -NR in the moiety of formula (I) 1 R 2 R 3 has a formula selected from the following: [ka] In some embodiments, the RAB portion has a structure selected from the following: [ka]

[0025] In some embodiments, the NAB is a DNA binding moiety ("DAB moiety"). In some embodiments, the DAB moiety is a groove binding moiety, an intercalating moiety, or a mixed mode binding moiety. In some embodiments, the DAB moiety comprises a bibenzimidazole moiety or a phenylphenanthridium moiety. In some embodiments, the DAB moiety has a structure selected from the following: [ka]

[0026] 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. In some embodiments, the NAM moiety has a structure selected from the following: [ka]

[0027] In some embodiments, the LDCD portion comprises at least one charged moiety. In some embodiments, the LDCD portion comprises at least one quaternary ammonium group. In some embodiments, the LDCD portion comprises at least one poly(ethylene glycol) moiety. In some embodiments, the poly(ethylene glycol) moiety has the formula: -(CH2CH2O) n -, where n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the LDCD portion includes one or more of the following groups:

[0028] [ka]

[0029] In some embodiments, the LDCD moiety comprises one or more alkylene groups (-(CH) n -), ether groups (-O-), thioether groups (-S-), ester bonds (-C(O)O-), carbamate bonds (-OC(O)NH-), sulfonamide bonds (-S(O)NH-), and any combination thereof. In some embodiments, the LDCD moiety is a branched LDCD moiety. In some embodiments, the branched LDCD moiety includes one of the following branch point functional groups: [ka] In some embodiments, the branched LDCD portion comprises: [ka]

[0030] In some embodiments, the multifunctional compound comprises an affinity moiety. In some embodiments, the affinity moiety is selected from biotin, a peptide tag, and an epitope. In some embodiments, the multifunctional compound comprises the following structure: [ka]

[0031] In some embodiments, the polyfunctional compound comprises a conjugated moiety. In some embodiments, the conjugated moiety comprises a haloalkane. In some embodiments, the haloalkane has the structure -(CH2) n -Y, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and Y is F, Cl, Br, or I. In some embodiments, n is 6 and Y is Cl.

[0032] In some embodiments, provided herein are systems or kits comprising the multifunctional compounds herein that comprise an affinity moiety or a conjugation moiety. In some embodiments, the systems or kits further comprise a reverse transcriptase and / or a DNA replication enzyme, and one or more amplification reagents and / or primers. In some embodiments, the systems or kits further comprise an affinity agent or a conjugation agent.

[0033] In some embodiments, the compounds, methods, systems, kits, etc. described in U.S. Patent No. 17 / 464,476, which is incorporated by reference in its entirety, find use in the embodiments herein. [Brief description of the drawings]

[0034] [Figure 1] A representative live / dead differentiation assay that does not require a photoactivation step is shown. [Diagram 2]1 shows data from vPCR reactions performed in live and dead cells with several concentrations of the compound CS0775. [Diagram 3] Data are shown for vPCR reactions performed with two amplicon sizes in representative Gram-negative bacteria, varying DMSO concentrations in the presence of compound CS0775. [Figure 4] 1 shows data from vPCR reactions in representative bacterial strains in the presence of compound CS0775 using primers optimized for various amplicon sizes. [Diagram 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 vPCR reaction data performed in the presence of various concentrations of several compounds of the present disclosure in representative bacterial strains. [Figure 7] 1 shows data from vPCR reactions in the presence of compound CS0775, with samples containing various cell concentrations. [Figure 8] Data are shown from vPCR reactions in samples with various combinations of live and dead cell ratios in the presence of compound CS0775. [Figure 9] Shown is data from vPCR reactions performed with intact and heat-inactivated viral particles treated with compound CS0775. [Figure 10] 1 shows an exemplary viral capsid integrity assay using the multifunctional nucleic acid crosslinkers herein. [Figure 11] ΔCt values ​​for different treatment conditions and amplicon lengths in a SARS-CoV-2 viral infectivity assay. [Figure 12] 1 shows the results of an AAV capsid integrity assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Provided herein are multifunctional compounds that bind and modify nucleic acids by forming crosslinks between nucleic acid strands. In certain embodiments, the multifunctional nucleic acid binding / modifying compounds are selective for the nucleic acids of non-living cells and viruses (i.e., they are excluded from or degraded within living cells and viruses) and therefore find use in methods for detecting or distinguishing living cells / viruses and nucleic acids therefrom.

[0036] The multifunctional compounds herein include nucleic acid binding moieties (e.g., DNA-selective binding moieties, RNA-selective binding moieties, non-selective nucleic acid binding moieties, etc.), nucleic acid modifying moieties, and viability differentiation motifs for distinguishing nucleic acids (e.g., DNA and / or RNA) associated with live cells from those associated with non-live cells. The method does not require a photoactivation step and allows for culture-independent detection of DNA from live cells with minimal interference from DNA of dead cells. The multifunctional compounds, compositions, kits, and methods can simplify the vPCR process and improve the consistency and robustness of viability tests based on molecular detection. The multifunctional compounds can also be used for other applications, such as methods for removing nucleic acids from a sample. In some embodiments, the multifunctional compounds further include affinity moieties and / or conjugation moieties for isolating or removing from a sample the nucleic acids bound by the multifunctional compounds herein.

[0037] In some embodiments, the multifunctional compounds herein contain an RNA binding moiety and are useful in multiple applications, including, but not limited to, determining viral capsid integrity as a surrogate for viral infectivity, determining the amount of mRNA or RNA encapsulated within lipid nanoparticles / exosomes or other delivery vectors, and removing unwanted RNA from a sample.

[0038] Many human or animal viral pathogens have RNA genomes. Viral infectivity assays often require BSL3 facilities. Such facilities are not very common. Simple methods with high sensitivity and specificity are required as alternatives. For example, many patients who are hospitalized with COVID-19 have been documented to have high viral loads. Although patients recover and are non-infectious with no culturable virus, nasal samples are PCR positive even weeks or months after recovery. This results in an increased burden on the healthcare system. If there was a PCR-based molecular system that was highly specific and sensitive and could also give information on whether the amplification product was from a membrane-compromised virus (non-infectious), it would help in the management of patients in the healthcare system. This could be applicable not only to SARS-CoV-2 but also to many pathogens.

[0039] In vitro transcribed RNA or mRNA encapsulated in lipid nanoparticles (or other delivery systems including but not limited to exosomes) can be used for the production of mRNA vaccines or for use as RNA transfection in life science laboratories. It is highly desirable to know how much RNA / mRNA is actually incorporated into lipid nanoparticles and how much is not. This will help in designing better lipid nanoparticles, optimizing experimental conditions, etc.

[0040] In many situations, it may be desirable to remove certain classes of RNA. One such example is the removal of globin mRNA from blood. Globin mRNA is highly abundant and often interferes with downstream applications such as next generation sequencing, array-based hybridization analysis, qPCR and dPCR, so it is highly desirable to remove globin RNA. Enzymatic methods using RNase H can be taxing on samples, and non-enzymatic methods for removing globin mRNA are not ideal. The compounds of the present invention can be used to simultaneously crosslink globin mRNA released from selective lysis of red blood cells. If the crosslinking molecule also has an affinity tag (e.g., biotin) or a conjugation tag (e.g., haloalkane), an affinity resin (streptavidin-coated beads) or a conjugation agent (e.g., HALOTAG) can be used to remove the crosslinked free globin RNA.

[0041] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0042] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, this document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein may 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.

[0043] Definitions of certain functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are identified in accordance with the Periodic Table of the Elements, inside cover of the CAS version of the Handbook of Chemistry and Physics, 75th Ed., and certain functional groups are generally defined as set forth therein. Additionally, general principles of organic chemistry, and specific functional groups and reactivities, are described in Sorrell, Organic Chemistry, 2nd edition, University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7th Edition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rd Edition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0044] 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 acyls include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, benzoyl, and the like.

[0045] 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.

[0046] As used herein, the term "alkyl" refers to an alkyl group having 1 to 30 carbon atoms, such as 1 to 16 carbon atoms (C 16 Alkyl), 1 to 14 carbon atoms (C1 to C 14 Alkyl), 1 to 12 carbon atoms (C1 to C 12 Alkyl), 1 to 10 carbon atoms (C1 to C 10 alkyl), 1 to 8 carbon atoms (C1 to C8 alkyl), 1 to 6 carbon atoms (C1 to C6 alkyl), 1 to 4 carbon atoms (C1 to C4 alkyl), 6 to 20 carbon atoms (C6 to C 20 alkyl), or 8 to 14 carbon atoms (C8 to C 14 "alkyl" refers to a straight or branched saturated hydrocarbon chain containing the radicals aryl, aryl, aryl, aryl and arylalkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0047] As used herein, the term "alkylene" refers to a straight or branched chain hydrocarbon having 1 to 10 carbon atoms (C1-C 10 It refers to a divalent group derived from a straight or branched chain hydrocarbon having 1 to 6 carbon atoms (C1-C6 alkylene), for example. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, and -CH(CH3)CH2CH2CH2CH2-.

[0048] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain 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.

[0049] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain 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.

[0050] As used herein, the term "aryl" refers to an aromatic carbocyclic ring system having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic), including fused ring systems, and no heteroatoms. As used herein, aryl refers to an aromatic carbocyclic ring system having 6 to 20 carbon atoms (C6-C 20 Aryl), 6 to 14 ring carbon atoms (C6-C 14 Aryl), 6 to 12 ring carbon atoms (C6-C 12 aryl), or 6 to 10 ring carbon atoms (C6-C 10 Representative examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like.

[0051] As used herein, the term "arylene" 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).

[0052] As used herein, the term "cycloalkyl" 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 cycloalkyl 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.

[0053] 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 from 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0054] As used herein, the term “halogen” or “halo” means F, Cl, Br, or I.

[0055] As used herein, the term "haloalkyl" means an alkyl group, as defined herein, 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.

[0056] As used herein, the term "heteroalkyl" refers to an alkyl group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently substituted with a heteroatom group, such as -NR-, -O-, -S-, -S(O)-, -S(O)2-, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which may be optionally substituted. As an example, one, two, or three carbon atoms may be independently substituted with the same or different heteroatom groups. Examples of heteroalkyl groups include, but are not limited to, -OCH3, -CHOCH3, -SCH3, -CHSCH3, -NRCH3, and -CHNRCH3, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may be optionally substituted. Heteroalkyl also includes groups in which the carbon atoms of the alkyl are oxidized (i.e., -C(O)-).

[0057] As used herein, the term "heteroalkylene" refers to an alkylene group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group, such as -NR-, -O-, -S-, -S(O)-, -S(O)2-, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which may be optionally substituted. As an example, one, two, or three carbon atoms may be independently replaced with the same or different heteroatom groups. Heteroalkylene also includes groups in which the carbon atoms of the alkyl are oxidized (i.e., -C(O)-). Examples of heteroalkylene groups include, but are not limited to, -CH2-O-CH2-, -CH2-S-CH2-, -CH2-NR-CH2-, -CH2-NH-C(O)-CH2, and the like, as well as polyethylene oxide chains, polypropylene oxide chains, and polyethyleneimine chains.

[0058] As used herein, the term "heteroaryl" 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 an appended monocyclic heteroaryl ring fused to a monocyclic aryl group as defined herein, or 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 heteroaryls 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, isobenzofuranyl, isoindoyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl.Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl. 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 "heterocycle" or "heterocyclic" refers to a saturated or partially unsaturated non-aromatic cyclic group having one or more ring heteroatoms independently selected from O, N, and S, meaning 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 0, 1, 2, or 3 double bonds and 1, 2, or 3 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, piperidinyl. , pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (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 joined 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. Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are joined 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, azaadamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), oxaadamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane). The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0060] As used herein, the term "hydroxy" refers to an --OH group.

[0061] 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 minimum number of carbon atoms in the group and y is the maximum number. Thus, for example, "C1-C3-alkyl" refers to an alkyl group containing from 1 to 3 carbon atoms.

[0062] As used herein, the term "substituent" refers to a group substituted on an atom of a designated group.

[0063] Where a group or moiety can be substituted, the term "substituted" indicates 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 indicated in the phrase using "substituted" can be replaced with a selection of the indicated groups listed, or with suitable groups known to those of 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.

[0064] For the compounds described herein, groups and substituents thereof may be selected according to the permissible valences of atoms and substituents such that their selection and substitution result in stable compounds, e.g., do not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like.

[0065] Where substituents are designated in a conventional chemical formula written from left to right, they optionally include the substituents resulting from writing the structure from right to left, e.g., -CHO- optionally also recites -OCH-, and -OC(O)NH- optionally also recites -NHC(O)O-.

[0066] In the present specification, the numerical ranges are expressly intended to include each intervening numerical value with the same degree of precision. For example, in the range of 6 to 9, the numerical values ​​7 and 8 are expressly intended in addition to 6 and 9, and in the range of 6.0 to 7.0, the numerical values ​​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 intended.

[0067] As used herein, "nucleic acid binding moiety" or "NAB moiety" refers to a moiety that interacts with nucleic acid non-covalently.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 intercalate between nucleotide base pairs), and mixed-mode NAB moieties (which include moieties that intercalate into the DNA double helix and moieties that protrude into grooves such as the minor groove).NAB moieties may bind to DNA, RNA, or both.

[0068] As used herein, a "DNA binding moiety" or "DAB moiety" is a moiety that specifically interacts with deoxyribonucleic acid (preferentially over RNA) in a non-covalent manner. In some embodiments, the DAB moiety exhibits at least a 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 100-fold, 200-fold, 500-fold, 100-fold, or more, or any range therebetween) increased affinity for DNA over RNA.

[0069] As used herein, an "RNA binding moiety" or "RAB moiety" is a moiety that specifically interacts with ribonucleic acid (preferentially over DNA) in a non-covalent manner. In some embodiments, the RAB moiety exhibits at least a 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 100-fold, 200-fold, 500-fold, 100-fold, or more, or any range therebetween) increased affinity for RNA over DNA.

[0070] 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 nucleobase, or can react with two different nucleobases to form a crosslink within the same strand (intrastrand) or between opposing strands (interstrand).

[0071] As used herein, the term "affinity moiety" refers to a functional group capable of forming a stable non-covalent interaction with an "affinity agent." Examples of affinity moieties include, but are not limited to, biotin and digoxigenin.

[0072] As used herein, the term "conjugating moiety" refers to a functional group capable of forming a covalent bond with a "conjugating agent." Exemplary conjugating moieties and conjugating agents are chloroalkanes and HALOTAG.

[0073] As used herein, the term "cell-permeable" refers to a compound or moiety that can effectively permeate the cell membrane of a non-viable cell, or the cell membrane of a viable cell that has been synthetically permeabilized, or the intact cell membrane of a cell, whether viable or non-viable. As used herein, the term "cell-impermeable" refers to a compound or moiety that cannot effectively pass the cell membrane of a viable cell that has not been synthetically permeabilized.

[0074] As used herein, the term "sample" is used in the broadest sense. It is primarily meant to include biological samples, food samples, and environmental samples, as well as specimens or cultures obtained from any source. Biological samples may be obtained from animals (including humans) and include liquids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Samples may also refer to cell lysates, including cells lysed with a lysing agent, or lysates such as rabbit reticulocyte lysate or wheat germ lysate. Samples may also include cell-free expression systems. Environmental samples include environmental materials such as surface materials, soil, water, crystals, and industrial samples. Such examples are not to be construed as limiting the types of samples applicable to the present disclosure.

[0075] As used herein, the term "microorganism" refers to any microscopic organism, including bacteria, actinomycetes, cyanobacteria (single-celled algae), fungi, protozoa, as well as viruses.

[0076] As used herein, the term "substantially" means that the recited properties, parameters, and / or values ​​need not be achieved exactly, but that deviations or variations, including, for example, tolerances, measurement errors, limits of measurement precision, and other factors known to those of skill in the art, may occur in amounts that do not interfere with the effect that the property is intended to provide. A substantially absent property or characteristic may be within the noise, background, detection capabilities of the assay used, or a small fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%) of a significant property.

[0077] 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 are well known and commonly used in the art. The meaning and scope of the terms should be clear, except that in the event of potential ambiguity, the 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.

[0078] 2.Compound The present disclosure includes compounds that can selectively bind and modify nucleic acid in non-viable cells without the need for a photoactivation step. Such compounds can be used in methods for selectively detecting nucleic acid from viable cells and for removing nucleic acid from a sample.

[0079] In one aspect, the present disclosure provides a multifunctional compound or a salt thereof, the multifunctional compound comprising: (A) Nucleic acid binding portion (“NAB portion”); (B) live cell / dead cell differentiation portion ("LDCD portion"); and (C) Nucleic acid modification moiety (“NAM moiety”).

[0080] In a second aspect, the disclosure provides a compound or a salt thereof, the multifunctional compound comprising: (A) Nucleic acid binding portion (“NAB portion”); (B) live cell / dead cell differentiation portion ("LDCD portion"); and (C) Affinity moiety.

[0081] In a third aspect, the disclosure provides a compound or a salt thereof, the multifunctional compound comprising: (A) Nucleic acid binding portion (“NAB portion”); (B) live cell / dead cell differentiation portion ("LDCD portion"); and (C) Conjugated part.

[0082] In a fourth aspect, the disclosure provides a compound or a salt thereof, the multifunctional compound comprising: (A) Nucleic acid binding portion (“NAB portion”); (B) Live cell / dead cell differentiation portion (“LDCD portion”); (C) a nucleic acid modification moiety (“NAM moiety”); and (D) Affinity or conjugation moieties.

[0083] In some embodiments, a multifunctional compound includes more than one NAB moiety (e.g., two NAB moieties), more than one LDCD moiety (e.g., two LDCD moieties), more than one NAM moiety (e.g., two NAM moieties), more than one conjugate moiety, and / or more than one affinity moiety. In some embodiments, a multifunctional compound includes one or more NAB moieties (e.g., two NAB moieties). In some embodiments, a multifunctional compound includes more than one NAM moiety (e.g., two NAM moieties). In some embodiments, a NAB moiety is a DAB moiety. In some embodiments, a NAB moiety is a RAB moiety.

[0084] In some embodiments, the multifunctional compound has a structure selected from the following: ABC, BAC, ACB, BCA, [ka] or AB(D)-C, where D is a substituent off of B; where A is a NAB moiety (e.g., a RAB or DAB moiety); B is an LDCD moiety; and C is a NAM moiety, affinity moiety, or conjugate moiety. When D is present, C is a NAM moiety and D is an affinity moiety or conjugate moiety.

[0085] In some embodiments, the polyfunctional compound has the structure ABC, where A is a NAB moiety, B is a LDCD moiety, and C is a NAM moiety. In such embodiments, the NAB moiety A is attached to the LDCD moiety B via a covalent bond, and the NAM moiety C is attached to the LDCD moiety B by another covalent bond.

[0086] In some embodiments, the polyfunctional compound has the structure ABC, where A is a NAB moiety, B is a LDCD moiety, and C is an affinity moiety. In such embodiments, the NAB moiety A is attached to the LDCD moiety B via a covalent bond, and the affinity moiety C is attached to the LDCD moiety B by another covalent bond.

[0087] In some embodiments, the polyfunctional compound has the structure ABC, where A is a NAB moiety, B is a LDCD moiety, and C is a conjugate moiety. In such embodiments, the NAB moiety A is attached to the LDCD moiety B via a covalent bond, and the conjugate moiety C is attached to the LDCD moiety B by another covalent bond.

[0088] In some embodiments, the polyfunctional compound has the structure AB(D)-C, where A is a NAB moiety, B is a LDCD moiety, C is a NAM moiety, and D is a conjugate moiety. In such embodiments, the NAB moiety A is attached to the LDCD moiety B via a covalent bond, the NAM moiety C is attached to the LDCD moiety B by another covalent bond, and the conjugate moiety D is attached to the LDCD moiety B via a covalent bond.

[0089] In some embodiments, the polyfunctional compound has the structure AB(D)-C, where A is a NAB moiety, B is a LDCD moiety, C is a NAM moiety, and D is an affinity moiety. In such embodiments, the NAB moiety A is attached to the LDCD moiety B via a covalent bond, the NAM moiety C is attached to the LDCD moiety B by another covalent bond, and the affinity moiety D is attached to the LDCD moiety B via a covalent bond.

[0090] The multifunctional compound comprises at least one NAB moiety (e.g., a RAB or DAB moiety), which is a moiety that interacts non-covalently with nucleic acids. The NAB moiety binds efficiently to nucleic acid species, facilitating rapid covalent modification of the nucleic acid by the NAM moiety. In some embodiments, the NAB moiety is a DAB moiety that interacts with DNA by binding to the major groove of the DNA double helix. In some embodiments, the NAB moiety is a DAB moiety that 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. In some embodiments, the NAB moiety is a RAB moiety that interacts with RNA.

[0091] Suitable NAB moieties include groups such as acridine, phenanthridine (e.g., phenylphenanthridium), dipyridine, terpyridine, phenanthroline, indole, quinoline, cyanine, quinacrine, benzothiazole, benzimidazole (e.g., bibenzimidazole), pyridocarbazole (e.g., pyridocarbazole dimer), aminoglycoside, and the like. In addition, a wide variety of nucleic acid stains are known, any of which (or a portion thereof) may be used as the NAB moiety of the multifunctional 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, 11th Edition (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 intercalating agents, such as ruthenium and rhodium complexes with ligands such as bipyridine, phenanthroline, 4,4,-diphenylbipyridine and their derivatives.

[0092] Representative examples of NAB moieties that can be used in the polyfunctional compounds described herein include the following: RNA-binding moieties, such as those of formula (I): [ka] or a tautomer or salt thereof (In the formula, R 1 and R 2 are each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, and aryl-C1-C6 alkyl, each of which is optionally substituted with 1 to 3 substituents, or R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl or heteroaryl ring; and R 3 is absent or is C1-C6 alkyl. In some embodiments, R 1 and R 2 are each independently selected from C1-C6 alkyl, and R 3 is absent or is C1-C6 alkyl. In some embodiments, R 1 , R 2 and R 3 are each independently selected from C1-C6 alkyl (e.g., methyl or ethyl). 1 and R 2 are each independently selected from C1-C6 alkyl (e.g., methyl or ethyl), and R 3 is absent. In some embodiments, R 3 does not exist, and R 1 and R 2together with the nitrogen atom to which they are attached form an optionally substituted, monocyclic 5- or 6-membered heterocyclyl or heteroaryl ring having 1, 2, or 3 heteroatoms independently selected from N, O, and S. In some embodiments, R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form a pyrrolidinyl, piperidinyl, morpholino, piperazinyl, or imidazolyl ring, each of which is optionally substituted with one substituent (e.g., C1-C6 alkyl, such as methyl). In some embodiments, the group -NR in the moiety of formula (I) 1 R 2 R 3 has a formula selected from the following: [ka]

[0093] Example RAB parts include: [ka]

[0094] Single-stranded DNA / RNA binding moieties, such as: [ka] where 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);

[0095] Inserts, such as: [ka]

[0096] Minor groove binders, such as: [ka] where 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);

[0097] Major groove binders, such as: [ka] (In the formula, R x is -CH2NH2 or H).

[0098] In certain embodiments, the NAB portion is selected from: [ka]

[0099] The multifunctional compounds include an LDCD moiety, which contains at least one LDCD motif that allows the multifunctional compound to bind and modify the DNA of dead cells, but not the DNA of living cells. The LDCD motif prevents the molecule from entering the living cell, or is processed by the living cell, preventing the multifunctional compound from modifying the DNA of living cells. The LDCD motif distinguishes the multifunctional compounds described herein from other compounds that contain NAB and NAM moieties (e.g., anti-cancer or research drugs, in which the multifunctional compounds are designed to be cell-permeable in order to bind and modify nucleic acids in living cells). In addition to at least one LDCD motif, the LDCD moiety can contain an alkylene group (-(CH2) nThe aryl group may contain other atoms or groups including, but not limited to, -), ether groups (-O-), thioether groups (-S-), amide bonds (-C(O)NH-), ester bonds (-C(O)O-), carbamate bonds (-OC(O)NH-), sulfonamide bonds (-S(O)NH-), phenylene bonds (-CH-), and any combination thereof. Additionally, any substitutable atom or group may be substituted with a suitable substituent (e.g., an amide bond may contain the group -C(O)NR-, where R is a suitable substituent, or a phenylene bond may contain one or more substituents on the phenyl group).

[0100] In some embodiments, the LDCD portion functions as a linker between one or more NAB portions and one or more NAM portions.

[0101] In some embodiments, the LDCD portion comprises at least one LDCD motif selected from a charged moiety, a high molecular weight moiety, an immobilization moiety, and a metabolically cleavable moiety.

[0102] In some embodiments, the LDCD motif is a charged moiety that prevents 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 one or more charged moieties. For example, in some embodiments, the LDCD moiety comprises one or more carboxylate groups, one or more sulfate groups, one or more sulfonate groups, one or more phosphate groups, or one or more ammonium groups (e.g., one 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 comprises at least one group of the formula -N +(CH3)2 groups. In some embodiments, the LDCD moiety comprises at least one carboxylate group. In some embodiments, the LDCD moiety comprises at least one group of the formula -CH2COO - or -CH2CH2COO - In some embodiments, the LDCD moiety comprises at least one phosphonate group. In some embodiments, the LDCD moiety comprises at least one group of the formula -CH2PO3 2- or CH2CH2PO3 2- The group includes:

[0103] In some embodiments, the LDCD motif is a high molecular weight moiety, e.g., a moiety having a molecular weight such that the molecular weight of the entire compound is 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. Compounds with high molecular weights may slow down mobility and affect cell permeability. The high molecular weight moiety may 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 prevent the polyfunctional compound from entering living cells (e.g., such that the entire compound has a molecular weight of 600 g / mol or more). 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, e.g., a peptide that has the property of rendering the molecule cell impermeable and not interfering with DNA binding.

[0104] In some embodiments, the LDCD motif is a metabolically cleavable moiety. Molecules with such moieties may be able to enter both living and non-living cells, but living cells will process the moiety to release the NAM moiety, thus preventing covalent modification. Since non-living cells cannot process the moiety, the NAM moiety can effectively interact with nucleic acids to prevent their amplification. For example, the trimethylquinone lock is a metabolically cleavable moiety that, when incorporated into a compound at the appropriate position, can be processed in living cells to release the NAB or NAM moiety, preventing the multifunctional compound from targeting and modifying DNA. For example, in some embodiments, in living cells, the trimethylquinone moiety is reduced by intracellular reductases. The resulting trimethylhydroquinone moiety undergoes a lactonization reaction to produce dihydrocoumarin. Further rearrangement of the remaining parts of the molecule then releases the NAM and / or NAB moieties. See, for example, WO2015 / 116867, and Mustafa et al. Bioconjugate Chem. 2016, 27, 1, 87-101. Each of these is incorporated herein by reference in its entirety. An exemplary reaction is shown in Scheme 1 below. Scheme 1. Metabolic cleavage of compounds containing the trimethylquinone-locked LDCD motif [ka]

[0105] In some embodiments, the LDCD motif comprises a solid support. When the multifunctional compound is bound to the solid support, a sample containing live and non-live cells can be mixed with the solid support such that DNA from the non-live cells binds to the solid support, but not the live cells. By separating the solid support from the rest of the sample, a sample containing only live cells is obtained, which can be used in a subsequent amplification reaction. This embodiment can also remove free DNA from other types of samples, and can be used in applications that require the generation of DNA-free reagents and / or solutions. The solid support can be, for example, beads, resins, magnetic particles, membranes, gels, ionic liquids (see, for example, 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.

[0106] In some embodiments, the LDCD motif is selected from the following group: [ka] and an alkylene group (-(CH2) n -), ether groups (-O-), thioether groups (-S-), ester bonds (-C(O)O-), carbamate bonds (-OC(O)NH-), sulfonamide bonds (-S(O)NH-), and any combinations thereof. Exemplary LDCD moieties include, but are not limited to, the following: [ka]

[0107] In some embodiments, the LDCD is branched, allowing for three or more other moieties (e.g., a NAB moiety, a NAM moiety, and an affinity or conjugation moiety) to be attached via the LDCD. For example, the LDCD may contain, in addition to the groups previously mentioned that comprise the LDCD, one of the following functional groups: [ka] An exemplary branched LDCD that finds use in embodiments herein is the following: [ka] wherein three moieties as described herein are attached thereto (e.g., one or more NAB moieties, one or more NAM moieties, one or more affinity moieties, one or more conjugate moieties (e.g., a NAM moiety, a NAB moiety, and an affinity moiety or conjugate moiety)). Exemplary compounds within the scope of the present specification that include a NAM moiety, a NAB moiety, an affinity moiety, and a branched LDCD are the following: [ka] The compounds herein may contain two or more branch points, allowing for the attachment of three or more moieties (e.g., 4, 5, 6, 7, 8, 9, 10, or more).

[0108] The multifunctional compound also includes at least one NAM moiety, which serves to covalently modify a nucleic acid and render it non-amplifiable. Exemplary NAM moieties include DNA alkylating agents, such as 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), alkylsulfonates (e.g., busulfan), seco-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 multifunctional compounds described herein.

[0109] Other NAM moieties include platinum-based moieties that modify DNA through the binding (via a coordinate covalent bond) of DNA nucleobases (typically via the N7 position of guanine residues) 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 of the NAM moiety in the multifunctional compounds described herein. For example, the multifunctional compounds may feature monodentate or bidentate ligands (e.g., moieties with one or more primary and / or secondary amines, and / or another source of coordinate nitrogen atoms, such as pyridine, quinoline, or phenanthridine moieties) that bind to the platinum center.

[0110] In some embodiments, the NAM moiety comprises a group selected from the following: [ka]

[0111] In some embodiments, the multifunctional compound is selected from the following or any salt thereof: [ka] [ka] [ka] [ka]

[0112] In some embodiments, the polyfunctional compounds herein include an affinity moiety. Such a moiety allows the polyfunctional compound to be non-covalently bound by a corresponding affinity agent. Exemplary affinity moieties that find use in the compounds herein include biotin, a peptide tag (e.g., 5xHis(HHHHH) (SEQ ID NO: 1), 6xHis(HHHHHH) (SEQ ID NO: 2), C-myc(EQKLISEEDL) (SEQ ID NO: 3), Flag(DYKDDDDK) (SEQ ID NO: 4), SteptTag(WSHPQFEK) (SEQ ID NO: 5), HA Tag(YPYDVPDYA) (SEQ ID NO: 6)), or an epitope. In some embodiments, a system is provided that includes a compound herein that includes (1) an affinity agent and (2) a corresponding affinity moiety. Suitable affinity moiety and affinity agent pairs for use in the embodiments herein include biotin and streptavidin, His tag and a metal ion (e.g., Ni 2+ ), FALG tag and antibodies.

[0113] In some embodiments, the polyfunctional compounds herein include a conjugation moiety. Such a moiety allows the polyfunctional compound to be covalently linked by a corresponding conjugation agent. Exemplary conjugation moieties that find use in the compounds herein include haloalkanes (e.g., coroalkanes). In some embodiments, the haloalkane moiety can be covalently linked by HALOTAG (Promega Corp.) or other dehalogenase proteins that have been modified to form a stable (e.g., covalent) bond (e.g., ester bond) with the haloalkyl substrate rather than releasing the halogenated substrate. In some embodiments, the haloalkanes that find use herein as conjugation moieties have the structure -(CH2) n In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and Y is a halogen (i.e., F, Cl, Br, or I). In some embodiments, n is 4, 5, 6, 7, or 8, and Y is Cl. In some embodiments, n is 6, Y is Cl, and Q has the formula -(CH2)6-Cl. Conjugated moieties can also be esters (-C(O)O-), amides (-C(O)NH-), carbamates (-NHC(O)O-), ureas (-NHC(O)NH-), phenylenes (e.g., 1,4-phenylene), linear or branched alkylenes, and / or oligoethylene glycols and polyethylene glycols (-(CH2CHO) x Linker moieties may also include various combinations of groups to provide linkers having a -O(CH2CH2O) bond, etc. In some embodiments, the linker has the formula -O(CH2CH2O) z1 -C(O)NH-(CH2CH2O) z2 -C(O)NH-(CH2) z3 -(OCH2CH2) z4 O-, where z1, z2, z3, and z4 are each independently selected from 0, 1, 2, 3, 4, 5, and 6. In some embodiments, a system is provided that includes the compound herein, which includes (1) a modified dehalogenase conjugator, and (2) a haloalkane conjugate moiety.

[0114] In some embodiments, suitable conjugate moiety and conjugate agent pairs utilize Staudinger ligation, amide coupling, methods using activated esters, imine bond formation (with and without ortho-boronic acids), boronic acid / diol interactions, disulfide bond formation, copper azide / copper-free azide, diazo and tetrazine "click" chemistry, UV-promoted thiolene conjugation, diazirine photolabeling, Diels-Alder cycloaddition, metathesis reactions, Suzuki cross-coupling, thiazolidine (step-4) coupling, etc. In some embodiments, systems are provided that include the compounds herein that include (1) a conjugation agent and (2) a corresponding conjugate moiety.

[0115] The polyfunctional compound may be in the form of a salt. In some embodiments, the neutral form of the polyfunctional compound may 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 polyfunctional compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise for purposes of this disclosure, the salts are equivalent to the parent form of the polyfunctional compound.

[0116] In particular, the polyfunctional compound may have functional groups that are anionic or may be anionic (e.g., -COOH may have -COO - can be, -SO3H can be -SO3 - or -P(O)(OH)2 can be -PO3 2- (which may be the case), may form a salt 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, NH5, NH6, NH7, NH8, NH9, NH10, NH11, NH20, NH30, NH41, NH42, NH43, NH44, NH45, NH46, NH47, NH48, NH49, NH50, NH51, NH52, NH53, NH54, NH55, NH56, NH57, NH58, NH59, NH60, NH61, NH62, NH63, NH64, NH65, NH66, NH67, NH68, NH69, NH70, NH71, NH72, NH73, NH74, NH75, NH76, NH77, NH78 + ) and substituted ammonium ions (e.g., NH3R1 + , NH2R2+ , NHR3 + , and NR4 + ) are included, but are not limited to. Some suitable examples of substituted ammonium ions include 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 multifunctional compound is a sodium salt.

[0117] The polyfunctional compound has a cationic or cationic functional group (e.g., -NH2 is -NH3 + (which may be the case) may form a salt with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid. 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 polyfunctional compound is a halide salt, such as a chloride salt, a bromide salt, an iodide salt, etc. In some embodiments, the polyfunctional compound is a tetrafluoroborate salt or a trifluoromethanesulfonate salt.

[0118] The polyfunctional compounds can be prepared by various methods, including those shown in the examples. The polyfunctional compounds and intermediates herein may 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, with optional pretreatment with activated charcoal, thin layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, for example, as described in "Vogel's Textbook of Practical Organic Chemistry", 5th edition (1989), Furniss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM20 2JE, England.

[0119] The reaction conditions and reaction times for each step may vary depending on the particular reactants used and the substituents present in the reactants used. Specific procedures are described in the Examples section. The reactions can be worked up in conventional manner, for example by removing the solvent from the residue and further purification according to methods commonly known in the art, such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise stated, 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 procedures selected from standard organic chemistry techniques, techniques analogous to the synthesis of known structurally similar compounds, or procedures analogous to those described in the Schemes or Synthetic Examples sections above.

[0120] Routine experimentation, including proper manipulation of reaction conditions, reagents and sequence of the synthetic route, protection of chemical functional groups that are incompatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the method, are within the scope of the present invention. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art, examples of which 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 polyfunctional compounds of the present invention can be accomplished by methods similar to those described above and in the synthetic schemes described in the specific examples.

[0121] 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 polyfunctional compounds or intermediates using standard procedures (chromatographic separation, recrystallization, enzymatic resolution, etc.).

[0122] Similarly, if a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the procedures described above using a pure geometric isomer as a starting material, or by resolving a mixture of geometric isomers of a multifunctional compound or intermediate using standard procedures such as chromatographic separation.

[0123] The synthetic methods and specific examples described are illustrative and should not be read as limiting the scope of the invention as defined in the claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are included within the scope of the claims.

[0124] 3. Compositions and Systems / Kits In some embodiments, the present disclosure provides compositions and systems / kits that include the multifunctional crosslinking compounds described herein (i.e., compounds that include at least a NAB portion, an LDCD portion, and a NAM portion).

[0125] Compositions comprising the multifunctional crosslinking compounds described herein may further comprise a solvent. In some embodiments, the solvent is water. In such embodiments, the composition may further comprise one or more water-soluble components, such as salts or buffers. In some embodiments, the solvent is an organic solvent, such as dimethyl sulfoxide (DMSO). Using DMSO as a solvent for the multifunctional compounds may provide additional advantages when amplifying DNA from samples containing Gram-negative bacteria, because such bacteria may further have a lipopolysaccharide layer that is more difficult for the multifunctional compounds to cross, even if the cells are non-viable. Using DMSO as a solvent may increase the DNA modification efficiency of samples containing Gram-negative bacteria.

[0126] 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 multifunctional compound alone or in a solvent, such as water or DMSO. When the multifunctional compound is provided alone, the system or kit may further comprise a solvent capable of dissolving the multifunctional compound. The system or kit may further comprise one or more reagents used to perform an amplification reaction, such as a viability PCR reaction or a viability RT-PCR reaction.

[0127] In some embodiments, the system or kit further comprises a DNA replicating enzyme.The DNA replicating enzyme that can be used according to these embodiments includes, but is not limited to, any replicating enzyme that can replicate DNA molecules.In some embodiments, the DNA replicating enzyme is a thermostable replicating enzyme, which is particularly useful in PCR applications.The thermostable replicating enzyme is isolated from a wide variety of thermophilic bacteria. Examples of such thermophilic bacteria include 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 Thermococcus species, Thermoplasma acidophilum (Tac), Thermotoga neapolitana (Tne), Thermotoga maritima (Tma) and other Thermotoga species, Pyrococcus furiosus (Pfu), Pyrococcus horikossii (Pho), Pyrococcus woesei (Pwo), Pyrococcus strain ES4 (ES4) and other Pyrococcus species, 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 mutants, variants, or derivatives thereof.Accordingly, in some embodiments, the system or kit comprises a thermostable DNA replicating enzyme 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 replicating enzyme is a Taq replicating enzyme. In other embodiments, the DNA replicating enzyme is a replicating enzyme with strand displacement activity, which is particularly useful in isothermal amplification reactions. DNA replicating enzymes with strand displacement activity are isolated from a wide variety of organisms. Such organisms include, for example, Bacillus smithii (Bsm), Bacillus stearothermophilus (Bst), Bacillus subtilis (Bsu), and Bacillus subtilis phage phi29 (phi29), as well as mutants, variants, or derivatives thereof. Accordingly, in some embodiments, the system or kit comprises a DNA replication enzyme selected from Bsm, Bst, Bsu, and phi29, or a mutant, variant, or derivative thereof.

[0128] In some embodiments, DNA replicating enzymes that may be used in accordance with these embodiments include, but are not limited to, commercially available DNA replicating enzymes (e.g., from 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).

[0129] In some embodiments, the system or kit further comprises a reverse transcriptase. In some embodiments, the reverse transcriptase may have endogenous RNase H activity. Endogenous RNase H activity is typically preferred in quantitative PCR applications because it enhances the melting of the RNA-DNA duplex during the first round of PCR. A variety of reverse transcriptases suitable for RT-qPCR are known in the art and may be used in the disclosed systems and methods. In some embodiments, M-MLV reverse transcriptase from Moloney murine leukemia virus or AMV reverse transcriptase from avian myeloblastosis virus is used in quantitative RT-PCR applications. M-MLV reverse transcriptase has weaker RNase H activity than AMV reverse transcriptase and is therefore the preferred reverse transcriptase for cDNA synthesis from long messenger RNA (mRNA) templates (>5 kb) (see, e.g., Mo et al., Methods Mol Biol., 926:99-112 (2012)). Thermostable RNase H-RTs have also been recently developed and may be used in conjunction with the systems and methods described herein.

[0130] In some embodiments, the system or kit further comprises one or more additional reagents, e.g., 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 to enable detection of the amplification, a buffer, a ligating enzyme, 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.

[0131] In some embodiments, the system or kit includes one or more primers. Generally, a primer is a short nucleic acid complementary to a long template. During replication, the primer may be extended based on the template sequence to produce a long nucleic acid that is a complementary copy of the template. Extension may occur, among others, by sequential addition of individual nucleotides (e.g., by the action of a replicating enzyme) or attachment of blocks of nucleotides (e.g., by the action of a ligase that joins a pair of primers). The primer may be DNA, RNA, an analog thereof (e.g., an artificial nucleic acid), or any combination thereof. The primer may have any suitable length, for example, 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. The primer is typically chemically synthesized. The primer may be provided as at least one pair of primers for the amplification of at least one nucleic acid target. The pair of primers may be a forward and reverse primer (ie, a sense and an antisense primer), which collectively define opposite ends (and thus the length) of the resulting amplicon.

[0132] In some embodiments, the system or kit provides a pair of primers that specifically detects a microorganism or cell of interest. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacterium is of 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 viral family selected from: Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also called 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 (such as 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., coronavirus); 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 virus);Bungaviridae (e.g., Hantavirus, Bungavirus, Phlebovirus, and Nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., Reovirus, Orbivirus, and Rotavirus); Birnaviridae; Hepadnaviridae (Hepatitis B viruses); Parvoviridae (Parvoviruses); Papovaviridae (Papillomaviruses, Polyomaviruses); Adenoviridae (Most Adenoviruses); Herpesviridae (Herpes Simplex Viruses ( HSV)-1 and HSV-2, Varicella-Zoster Virus, Cytomegalovirus (CMV), Herpes Virus; Poxyiridae (Variolavirus, Vaccinia Virus, Variola Virus); and Iridoviridae (African Swine Fever Virus, etc.); and unclassified viruses (e.g., etiological agents of spongiform encephalopathies, etiological agents of delta hepatitis (thought to be a defective satellite of hepatitis B virus), etiological agents of non-A, non-B hepatitis (Class 1 = internal infection; Class 2 = parent infection (e.g., hepatitis C); Norwalk virus and related viruses, and Astrovirus)). 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 albicans). In some embodiments, the cell of interest is a eukaryotic cell, such as a mammalian cell or a plant cell.;

[0133] Some embodiments herein find use in the detection of RNA from live cells and / or intact viruses. In such embodiments, compounds described herein having a RAB moiety are used to bind RNA in non-living cells and / or viruses (e.g., RNA viruses) with disassembled capsids. Amplification of remaining RNA (e.g., not cross-linked by the multifunctional compounds described herein) reveals RNA from live cells and / or viruses with intact capsids. In some embodiments, the virus is an RNA virus, such as one of the Nidovirales, Picornavirales, or Tymovirales. In some embodiments, the virus is an RNA virus such as those of the Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaaflexiviridae, Gammaflexiviridae, Tymoviridae, Caliciviridae, Flaviviridae, Togaviridae, etc. In some embodiments, the RNA virus is an important human pathogen such as SARS-CoV-2, HIV-1 rhinovirus, hepatitis A virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, Zika virus, etc.

[0134] The pair of primers can be designed to detect amplicons of a particular length. For example, the amplicons may be from about 200 base pairs to about 1000 base pairs, or from about 400 base pairs to about 750 base pairs. For example, in some embodiments, the primers in the system or kit are for amplicons 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 amplicons of about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 base pairs.

[0135] In some embodiments, the system or kit may also include one or more probes, or any nucleic acid bound to at least one label, such as at least one dye. The probe may be a sequence-specific binding partner to the nucleic acid target and / or amplicon. The probe may be designed to allow detection of the target amplification based on fluorescence resonance energy transfer (FRET), including one or more nucleic acids attached to a pair of dyes that collectively exhibit fluorescence resonance energy transfer (FRET) when in close proximity to each other. The dye pair may provide, among other things, a first and a second emitter, or an emitter and a quencher. The fluorescence emission from the dye pair changes when the dyes separate from each other, such as by cleavage of the probe during primer extension (e.g., 5' nuclease assays such as TAQMAN probes) or when the probe hybridizes to the amplicon (e.g., molecular beacon probes). The nucleic acid portion of the probe may have any suitable structure or origin. For example, this portion may be a locked nucleic acid, a member of a general probe library, etc. In other cases, the probe and one of the primers of the primer pair may be combined within the same molecule. For example, a primer molecule may contain a primer sequence at its 3' end and a molecular beacon-type probe at its 5' end. This arrangement 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 only one base (single nucleotide polymorphisms (SNPs)).

[0136] In some embodiments, the system or kit may also include one or more label or reporter molecules. Exemplary dyes used for labeling are fluorescent dyes (fluorophores) and fluorescent quenchers. Reporters include any compound or set of compounds that reports a state, such as the extent of a reaction. Exemplary reporters include at least one dye, such as a fluorescent dye or an 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.).

[0137] 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.

[0138] In some embodiments, the system or kit further comprises 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 replication enzyme) may be provided as a solution in the buffer. Exemplary buffers include tris(hydroxymethyl)aminomethane (Tris) buffer. The buffer may further comprise 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 specifically adapted for the DNA replication enzyme provided in 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.

[0139] In some embodiments, the system or kit further comprises a reverse transcriptase. The reverse transcriptase is used in a RT-PCR reaction to generate complementary DNA (cDNA) from RNA, and the cDNA can then be amplified in an amplification reaction. The RT-PCR reaction may be used to detect various RNA species, such as RNA from viruses. The system or kit may further comprise other components for RT-PCR, such as a ribonuclease inhibitor that inhibits target degradation during cDNA synthesis.

[0140] In some embodiments, kits are provided that include one or more or all of the components necessary, sufficient, or useful for carrying out the methods described herein (e.g., compounds described herein, DNA replicating enzymes, 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 shipping and storage of the kit.

[0141] 4.How to use Embodiments of the present disclosure include methods of amplifying nucleic acids (e.g., DNA or RNA) from a sample, where the nucleic acids (e.g., DNA or RNA) are selectively amplified from viable cells and not from non-viable cells in the sample (i.e., an amplification reaction such as a viability PCR or RT-PCR reaction).

[0142] Generally, an amplification reaction involves forming a replica or copy (e.g., a direct copy and / or a complementary copy) of a nucleic acid or a segment thereof. A replication reaction generally involves enzymes, such as replication enzymes and / or ligation enzymes, among others. The nucleic acid and / or segment to be replicated is the template (and / or target) for replication. The reaction also generally involves a process of amplification, i.e., replication occurs repeatedly over time to form multiple copies of at least one segment of the template molecule. As amplification proceeds, it may cause an exponential or linear increase in copy number. A typical amplification results in a greater than 1,000-fold increase in copy number and / or signal. Exemplary amplification reactions for the assays disclosed herein may include polymerase chain reaction (PCR) or ligation chain reaction (LCR), each of which is driven by thermal cycling. In some embodiments, the reaction involves reverse transcribing RNA from the sample into cDNA. The cDNA is then amplified using the methods described herein. Thermal cycling generally involves cycling the reaction mixture between heating and cooling to perform a series of denaturation (melting), annealing, and extension steps. Other exemplary amplification reactions include isothermal amplification methods that use enzymes with strand displacement activity (e.g., DNA replicating enzymes).

[0143] In some embodiments, the disclosure provides a method of detecting a viable microorganism or cell of interest in a sample, the method comprising: (a) contacting the sample with a compound described herein (i.e., a compound comprising a NAB portion, a LDCD portion, and a NAM portion) 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 acid from the second mixture to generate a detectable signal, where the signal indicates the presence of viable microorganisms in the sample. In some embodiments, the NAB is a RAB portion and amplifying the nucleic acid comprises reverse transcribing the RNA into cDNA and then amplifying the cDNA.

[0144] In some embodiments, the disclosure provides a method of amplifying nucleic acid from a sample, the method comprising: (a) contacting the sample with a compound described herein (i.e., a compound comprising a NAB portion, a LDCD portion, and a NAM portion) 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 acid from the second mixture. In some embodiments, the NAB is a RAB portion and amplifying the nucleic acid comprises reverse transcribing RNA into cDNA and then amplifying the cDNA.

[0145] In some embodiments, the present disclosure provides a method for detecting a viable microorganism or cell of interest in a sample, the method comprising: (a) contacting the sample with a compound described herein (i.e., a compound comprising a NAB portion, a LDCD portion, and a NAM portion) or a salt thereof to form a first mixture; (b) removing the multifunctional compound described herein (i.e., a compound comprising a NAB portion, a LDCD portion, and a NAM portion) from the first mixture to form a second mixture; and (c) amplifying nucleic acid from the second mixture to generate a detectable signal, where the signal indicates the presence of viable microorganisms in the sample. In some embodiments, the NAB is a RAB portion, and amplifying the nucleic acid comprises reverse transcribing the RNA into cDNA and then amplifying the cDNA.

[0146] In some embodiments, the disclosure provides a method of removing nucleic acid from a non-viable microorganism or cell of interest from a sample, the method comprising: (a) contacting the sample with a compound described herein (i.e., a compound comprising a NAB moiety, an LDCD moiety, and an affinity moiety (and optionally a NAM moiety)), or a salt thereof, to allow the multifunctional compound to bind to nucleic acid from the non-viable microorganism or cell of interest to form a first mixture; (b) removing nucleic acid from the non-viable microorganism or cell of interest from the first mixture by contacting the first mixture with an affinity agent, allowing the affinity agent to bind to the affinity moiety, and removing the affinity agent from the first mixture to form a second mixture; and (c) amplifying nucleic acid from the second mixture to generate a detectable signal, where the signal indicates the presence of viable microorganisms in the sample. In some embodiments, the NAB is a RAB moiety, and amplifying the nucleic acid comprises reverse transcribing the RNA into cDNA and then amplifying the cDNA. In some embodiments, the microorganism is an RNA virus.

[0147] In some embodiments, the disclosure provides a method of removing nucleic acid from a viable microorganism or cell of interest from a sample, the method comprising: (a) contacting the sample with a compound described herein (i.e., a compound comprising a NAB moiety, an LDCD moiety, and a conjugate moiety (and optionally a NAM moiety)), or a salt thereof, to allow the multifunctional compound to bind to nucleic acid from the non-viable microorganism or cell of interest to form a first mixture; (b) removing nucleic acid from the non-viable microorganism or cell of interest from the first mixture by contacting the first mixture with a conjugate agent, allowing the conjugate agent to bind to the conjugate moiety, and removing the conjugate agent from the first mixture to form a second mixture; and (c) amplifying the nucleic acid from the second mixture to generate a detectable signal, where the signal is indicative of the presence of viable microorganisms or cells in the sample. In some embodiments, the NAB is a RAB moiety and amplifying the nucleic acid comprises reverse transcribing the RNA into cDNA and then amplifying the cDNA. In some embodiments, the microorganism is an RNA virus.

[0148] As discussed herein, the multifunctional compounds disclosed herein do not require a photoactivation step to modify nucleic acids (e.g., DNA). This provides an advantage to viability amplification processes (e.g., PCR, RT-PCR, etc.) because it can reduce sample-to-sample variation, especially in complex or turbid samples. Thus, in some embodiments, the methods described herein do not include a photoactivation step.

[0149] Furthermore, the multifunctional compounds described herein can modify nucleic acids (e.g., DNA, RNA) from non-living cells in a sample, while the LDCD moiety prevents entry into or allows degradation of the LDCD in living cells or viruses. This allows selective labeling of nucleic acids (e.g., DNA, RNA) from non-living cells in a sample without the need for a culture step to increase the number of living cells in the sample. Thus, in some embodiments, the methods described herein do not include a culture step.

[0150] The sample in which the microorganism or cell is detected and / or the nucleic acid is amplified can be any sample in which it would be desirable to selectively amplify the nucleic acid from a living cell or to detect a living microorganism or cell in the sample. In some embodiments, the sample can be obtained from a 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 juice, 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, enhanced fluid, cavity fluid, sputum, pus, microflora, meconium, breast milk, and / or other excretions or biological tissues. The sample may 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, a pharmaceutical, a water sample, or a soil sample.

[0151] In some embodiments, the sample may be a sample suspected of containing viable microorganisms, for which it would be desirable to detect the presence of such viable microorganisms. For example, in some embodiments, the sample is a sample suspected of containing bacteria, such as bacteria from a genus selected from Actinomyces, Bacteroides, Bacillus, Bordetella, Campylobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Fusobacteria, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pseudomonas, Staphylococcus, Streptobacillus, Streptococcus, Treponema, Vibrio, and Yersinia. For example, in certain embodiments, the bacteria is Escherichia coli (e.g., E. 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 a sample suspected of containing a virus, such as a virus of 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 a sample suspected of containing a fungus, yeast, or another type of eukaryotic cell, such as a mammalian cell or a plant cell. In some embodiments, the sample is suspected of containing a virus. In some embodiments, the virus is an RNA virus, such as one of the Nidovirales, Picornavirales, or Tymovirales. In some embodiments, the virus is an RNA virus such as those of the Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaaflexiviridae, Gammaflexiviridae, Tymoviridae, Caliciviridae, Flaviviridae, Togaviridae, etc. In some embodiments, the RNA virus is an important human pathogen such as SARS-CoV-2, HIV-1 rhinovirus, hepatitis A virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, Zika virus, etc.

[0152] Some methods disclosed herein include contacting a sample with a multifunctional compound comprising a NAB moiety (e.g., a RAB moiety, a DAB moiety, etc.), a LDCD moiety, and a NAM moiety (and optionally an affinity moiety or conjugate moiety) to form a first mixture. In some embodiments, the methods herein include contacting a sample with a compound comprising a NAB moiety (e.g., a RAB moiety, a DAB moiety, etc.), a LDCD moiety, and an affinity or conjugate moiety (and optionally a NAM moiety). The contacting step may be performed by adding a multifunctional compound to the sample and incubating the sample for a time sufficient for the multifunctional compound to bind to and modify nucleic acids in the sample that are not present in live cells or intact viruses (i.e., free nucleic acids or nucleic acids in non-live cells, or disassembled capsid viruses). The contacting step can be carried out for about 5 minutes to about 180 minutes, or about 60 minutes to about 120 minutes, e.g., about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, or 120 minutes. In some embodiments, step (a) comprises contacting the sample with the multifunctional compound for about 90 minutes.

[0153] The concentration of the multifunctional compound in the first mixture can be in the range of about 1 micromolar to about 200 micromolar, or about 5 micromolar to about 100 micromolar, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 micromolar.

[0154] Step (a) can be carried out at any suitable temperature. For example, step (a) can be carried out at room temperature (e.g., about 20-25°C) or at about 37°C.

[0155] In some embodiments, the contacting step of step (a) is performed by adding a composition comprising a multifunctional compound, e.g., a multifunctional 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 dimethylsulfoxide (DMSO). The use of a solvent such as DMSO may be particularly advantageous in samples containing gram-negative bacteria, as discussed above.

[0156] In certain embodiments, step (b) of some methods herein involves removing, inactivating, or otherwise neutralizing the multifunctional 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 the NAM moieties in any excess compounds in the first mixture, thereby rendering them unable to modify further nucleic acids and ensuring optimal performance in the subsequent amplification reaction. However, in some embodiments, the NAM moieties that modified the nucleic acids in the sample are not affected by this step. For example, if excess compounds remain after step (a), the subsequent lysis of live cells in the sample will be altered by the excess compounds, making them unavailable for the amplification reaction. In such embodiments, the inactivating agent depends on the particular NAM moiety in the multifunctional 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 compound containing a thiol, 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 inactivating step serves to inhibit the NAB (e.g., DAB or RAB) moiety, thereby preventing the multifunctional compound from binding to and modifying DNA. In such embodiments, the inactivating agent depends on the particular NAB moiety in the multifunctional compound. In still other embodiments, the multifunctional compound is physically removed from the first mixture to form the second mixture, for example, by washing the sample.In embodiments in which the multifunctional compound comprises an affinity moiety or a conjugate moiety, the multifunctional compound in the first mixture (free or bound to nucleic acid) is bound by the affinity moiety or conjugate agent, and removal of the agent from the mixture provides an efficient step of removing unbound / unreacted multifunctional compound from the mixture.

[0157] In some embodiments, step (c) comprises amplifying nucleic acid from the second mixture to generate a detectable signal, where the signal indicates the presence of viable bacteria in the sample. The amplification reaction can be carried out in a variety of ways. For example, in some embodiments (e.g., when the target nucleic acid is DNA), the amplification step (c) comprises: (i) lysing cells and / or viruses in the second mixture to form a lysed sample; (ii) adding a DNA replicating enzyme 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 acid from the sample. In other embodiments, the amplification step (c) comprises: (i) lysing cells in the second mixture to form a lysed sample; (ii) adding a DNA replicating enzyme and an amplification reagent to the lysed sample to form a mixture; and (iii) subjecting the mixture to an isothermal amplification reaction to amplify nucleic acid from the sample.

[0158] In some embodiments, step (c) comprises reverse transcribing RNA from the second mixture to produce cDNA and then amplifying the cDNA to obtain a detectable signal, where the signal indicates the presence of a viable organism (e.g., an RNA virus) in the sample. The reverse transcription / amplification reaction can be performed in a variety of ways. For example, in some embodiments, step (c) comprises (i) lysing cells and / or viruses in the second mixture to form a lysed sample; (ii) adding a reverse transcriptase and an amplification reagent to the lysed sample to form a mixture; (iii) subjecting the mixture to a thermal cycling protocol to reverse transcribe the RNA into cDNA; (iv) adding a DNA replicating enzyme and an amplification reagent to the lysed sample to form a mixture; and (v) subjecting the mixture to a thermal cycling protocol to amplify nucleic acids from the sample. In some embodiments, steps (ii)-(iv) are performed simultaneously.

[0159] In some embodiments, the lysis step exposes nucleic acids (e.g., RNA and / or DNA) from live cells or viruses in the sample so that they can be amplified (and / or reverse transcribed) in an amplification reaction. Any suitable cell lysis method can be used, for example, chemical lysis, electrochemical lysis, acoustic lysis (i.e., sonication), mechanical lysis, or thermal lysis.

[0160] The methods herein may include additional steps and / or the order of steps may be altered according to the particular application. For example, in some embodiments, the method further includes removing contaminants and / or cellular debris from the lysed sample prior to adding the amplification enzymes and reagents. For example, this step may require purifying the nucleic acids (e.g., RNA and / or DNA) from the sample to generate a purified nucleic acid (e.g., RNA and / or DNA) sample to which the amplification enzymes and reagents can be added. The nucleic acids (e.g., RNA and / or DNA) can be purified using any conventional means, such as organic extraction followed by ethanol precipitation, salt-based precipitation, magnetic particle-based isolation, or solid phase adsorption. In certain embodiments, affinity moieties and / or conjugation moieties on the multifunctional compound are used for purification / isolation by contacting the nucleic acids bound by the multifunctional compound with an affinity or conjugation agent. In some embodiments, the affinity or conjugation agent is bound to a solid surface (e.g., beads, columns, plates, etc.). In some embodiments, unbound components of the sample (eg, nucleic acids in live cells or viruses that have not been bound / modified by a multifunctional compound) are separated from drug-bound nucleic acids by known methods.

[0161] In some embodiments where the nucleic acid to be analyzed / manipulated in the sample is DNA, the DNA is amplified in an amplification reaction such as PCR. In general, PCR includes any nucleic acid amplification reaction that relies on alternating cycles of heating and cooling (i.e., thermocycling) to achieve successive successive copies. PCR may be performed by thermocycling between two or more temperature set points, such as a high melting (denaturing) temperature and a low annealing / extension temperature, or between three or more temperature set points, such as a high melting temperature, a low annealing temperature, and an intermediate extension temperature. PCR generally results in an exponential increase in the amount of product amplicon over successive cycles.

[0162] In some embodiments where the nucleic acid to be analyzed / manipulated in a sample is RNA, the RNA may be reverse transcribed using a reverse transcriptase and amplification reagents in an RT-PCR reaction to first produce cDNA from the RNA, which may then be amplified using the methods and reagents described herein. Any of the amplification techniques or reagents described herein for amplifying DNA may be employed to amplify the cDNA reverse transcribed from the RNA.

[0163] Any suitable PCR technique or combination of techniques 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, exponential PCR followed by linear PCR, ligation-mediated PCR, methylation specific PCR, mini-primer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nested PCR, overlap extension PCR, replicase 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 combinations thereof, among others.

[0164] In some embodiments, nucleic acid (e.g., DNA or RNA) is amplified in an isothermal amplification reaction. Isothermal amplification reactions generally include 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 recombinant replicase amplification (RPA).

[0165] Amplification may be performed by adding an amplification reagent and a DNA replicating enzyme to form a mixture. DNA replicating enzymes that can be used according to these embodiments include, but are not limited to, any replicating enzyme that can replicate DNA molecules. In some embodiments, the DNA replicating enzyme is a thermostable replicating enzyme that is particularly useful in PCR applications. In some embodiments, the thermostable DNA replicating enzyme 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 replicating enzyme is a Taq replicating enzyme. In some embodiments, the DNA replicating enzyme is a replicating enzyme with strand displacement activity that is useful in isothermal amplification applications. In some embodiments, the DNA replicating enzyme with strand displacement activity is selected from Bsm, Bst, Bsu, and phi29 DNA replicating enzymes.

[0166] 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 to enable detection of the 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.

[0167] In some embodiments, the amplification reagent comprises 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, for example, 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. The primers are typically chemically synthesized. The primers can be provided as at least one pair of primers for the 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 opposing ends (and therefore the length) of the resulting amplicon.

[0168] In some embodiments, the amplification reagent comprises 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 of 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 viral 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 virus is an RNA virus, such as from the Nidovirales, Picornavirales, or Tymovirales.In some embodiments, the virus is an RNA virus, such as Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaaflexiviridae, Gammaflexiviridae, Tymoviridae, Caliciviridae, Flaviviridae, Togaviridae. In some embodiments, the RNA virus is an important human pathogen, such as SARS-CoV-2, HIV-1 rhinovirus, Hepatitis A virus, Yellow Fever virus, West Nile virus, Hepatitis C virus, Dengue virus, Zika virus, etc. In some embodiments, the microorganism is yeast. In some embodiments, the cell type of interest is a plant cell or a mammalian cell.

[0169] A pair of primers can be designed to detect an amplicon of a particular length. For example, the amplicon may 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.

[0170] In some embodiments, the amplification reagent comprises a magnesium salt, such as magnesium chloride or magnesium sulfate, hi some embodiments, the amplification reagent comprises magnesium chloride.

[0171] In some embodiments, the amplification reagent comprises a buffer. Exemplary buffers include tris(hydroxymethyl)aminomethane (Tris) buffer. The buffer may further comprise 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 replicating enzyme 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.

[0172] In some embodiments, such as embodiments of the method for detecting viable microorganisms in a sample, the amplification reagent comprises one or more probes, or any nucleic acid bound to at least one label, such as at least one dye. The probe may be a sequence-specific binding partner to the nucleic acid target and / or amplicon. The probe may be designed to allow detection of the amplification of the target based on fluorescence resonance energy transfer (FRET), including one or more nucleic acids attached to a pair of dyes that collectively exhibit fluorescence resonance energy transfer (FRET) when in close proximity to each other. The dye pair may provide, among other things, a first and a second emitter, or an emitter and a quencher. The fluorescence emission from the dye pair changes when the dyes separate from each other, such as by cleavage of the probe during primer extension (e.g., 5' nuclease assays such as TAQMAN probes) or when the probe hybridizes to the amplicon (e.g., molecular beacon probes). The nucleic acid portion of the probe may have any suitable structure or origin. For example, this portion may be a locked nucleic acid, a member of a general probe library, etc. In other cases, the probe and one primer of the primer pair may be bound in the same molecule. For example, a primer molecule may contain a primer sequence at its 3' end and a molecular beacon-type probe at its 5' end. This arrangement 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 only one base (single nucleotide polymorphisms (SNPs)).

[0173] In some embodiments, the amplification reagents also include one or more labels or reporter molecules. Exemplary dyes used for labeling are fluorescent dyes (fluorophores) and fluorescent quenchers. Reporters include any compound or set of compounds that reports a state, such as the extent of a reaction. Exemplary reporters include at least one dye, such as a fluorescent dye or an 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.).

[0174] In accordance with the embodiments provided herein, the concentrations of the amplification reagents described above may 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 the concentration ranges for the compounds of the present disclosure, will vary depending on the particular reaction conditions and reagents used, and are not meant to be limiting thereof.

[0175] In some embodiments, such as those involving PCR, the method further comprises heating the mixture to a temperature of at least 90° C. after adding the DNA replicating enzyme and the amplification reagents to form the mixture and before subjecting the mixture to a thermal cycling protocol to activate the DNA replicating enzyme. For example, the DNA replicating enzyme may not be initially reactive 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 about 2 minutes to about 5 minutes, e.g., about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

[0176] 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 to 68° C.; and (3) an extension step comprising subjecting the mixture to a temperature of about 50-72°C; wherein the series of steps (1) to (3) are repeated about 10 or more times in succession.

[0177] The denaturation step involves subjecting the mixture to a temperature of about 90-96° C. to denature the double-stranded DNA and allow for subsequent annealing of the primers. 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 carried out for about 15 seconds to about 60 seconds, e.g., about 15 seconds, about 30 seconds, about 45 seconds, or about 60 seconds.

[0178] The annealing step involves subjecting the mixture to a temperature of about 45°C to about 68°C to allow the primers to bind to their complementary sequences on the denatured DNA. The optimal annealing temperature depends on the particular 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 carried out for about 15 seconds to about 60 seconds, for example, about 15 seconds, about 30 seconds, about 45 seconds, or about 60 seconds.

[0179] The extension step involves subjecting the mixture to a temperature of about 50-72°C to allow the DNA replicating enzyme to extend the DNA strand starting from the annealed primer. The extension temperature depends on the DNA replicating enzyme used and the extension time depends on the length of the amplicon. Typical extension temperatures are about 50°C to about 72°C, such as 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. Typical extension times are 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.

[0180] The sequence of denaturation, 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.

[0181] 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 includes: (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 between 45 and 70°C; wherein the series of steps (1a) to (2a) is repeated 10 or more times in succession.

[0182] 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.

[0183] 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.

[0184] In some embodiments, the nucleic acid is amplified using an isothermal amplification protocol.

[0185] In a method of detecting viable microorganisms or living cells in a sample, the method generates a detectable signal, the signal being indicative of the presence of viable microorganisms or living cells in the sample. In some embodiments of such a method, the method further comprises detecting a detectable signal from the sample. The particular detection step depends on the probe and / or dye used in the method. Any suitable detection method may be used, such as photochemical, biochemical, spectroscopic, immunochemical, electrical, optical, or chemical means. In some embodiments, the detection step is a fluorescent detection step. For example, in some embodiments, the amplification product may be directly detected using fluorescence. In some embodiments, the fluorescent probe of the amplification product may be detected using fluorescence.

[0186] 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).

[0187] In some embodiments, the present disclosure provides a method of removing nucleic acids from a sample, the method 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 for any sample from which it is desirable to remove free nucleic acids. For example, certain reagents or solutions intended for use as human injections may be treated with a multifunctional compound described herein to remove free DNA.

[0188] 5. Working Example It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and recognizable, and may be made using suitable equivalents without departing from the scope of the 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 present disclosure, and should not be considered as limiting the scope of the disclosure. The disclosures of all journal articles, U.S. patents, and publications referred to herein are incorporated herein in their entirety by reference.

[0189] Example 1. Viability PCR workflow A diagram of the viability PCR (vPCR) workflow described in this application is shown in Figure 1. In this procedure, a live cell impermeable nucleic acid modifying compound is incubated with a mixture of bacterial cells, which enters only dead / dying cells due to the compromised permeability of the cell membrane. As a result, only nucleic acids from non-viable cells are covalently cross-linked, preventing their subsequent amplification by PCR. Thus, the signal observed in the vPCR detection assay comes exclusively from live cells. Dead cells are represented by a grey membrane with holes, and live cells by an intact purple membrane.

[0190] Example 2. Synthesis of compounds Abbreviations used in this example are as follows: 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.

[0191] Compound CS0729 [ka] 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 RT for 3 h and quenched by the addition of 2 mL of an aqueous solution of CH3CN / 0.1% TFA (1 / 1). 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.

[0192] 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) / TIPS (0.1 mL) and stirred for 30 min. The reaction was concentrated in vacuo and purified by reverse phase HPLC to give 5-(4-((2-(14-azanayl)ethyl)carbamoyl)benzyl)-3,8-diamino-6-phenylphenanthridin-5-ium 2,2,2-trifluoroacetate.

[0193] Step 3: 5-(4-((2-(l4-azanayl)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 RT for 3 h and quenched by adding 2 mL of an aqueous solution of CH3CN / 0.1% TFA (1 / 1). 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).

[0194] Compounds CS0727, CS0775, CS0776, CS0777, CS0935, and CS0942 were synthesized analogously to the procedure described above, and their structures and characterization data are shown in Table 1. [Table 1]

[0195] Compound CS0733 [ka] 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 RT for 3 h and quenched by adding 2 mL of an aqueous solution of CH3CN / 0.1% TFA (1 / 1). The mixture was then purified on silica gel 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.

[0196] Step 2: tert-Butyl (2-(5-(4-(5-(4-methylpiperazin-1-yl)-1H,3'H-[2,5'-bibenzo[d]imidazol]-2'-yl)phenoxy)pentanamide)ethyl)carbamate (67 mg, 0.1 mmol, 1.0 equiv) was dissolved in TFA (1 mL) / TIPS (0.1 mL) and stirred for 30 min. 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)pentanamide tetrakis(2,2,2-trifluoroacetate).

[0197] Step 3: N-(2-aminoethyl)-5-(4-(5-(4-methylpiperazin-1-yl)-1H,3'H-[2,5'-bibenzo[d]imidazol]-2'-yl)phenoxy)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), and DIPEA (117 mg, 0.9 mmol, 9.0 equiv) in DMF (2 mL). The solution was stirred at RT for 3 h and concentrated in vacuo. The mixture was purified on normal phase silica gel to give CS0733.

[0198] Compound CS0717 was synthesized analogously to the above procedure, and its structure and property data are shown in Table 2. [Table 2]

[0199] Compound CS0868 [ka] 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-amine tetrakis(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 RT 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).

[0200] 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)propanoic acid Tris(2,2,2-trifluoroacetate) (95 mg, 0.1 mmol, 1.0 equiv) was dissolved in TFA (1 mL) / TIPS (0.1 mL) and stirred for 30 min. The reaction was concentrated in vacuo and purified by reverse phase HPLC to give the 2,2,2-trifluoroacetate compound along with 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).

[0201] Step 3: N-(2-aminoethyl)-5-(4-(5-(4-methylpiperazin-1-yl)-1H,3'H-[2,5'-bibenzo[d]imidazol]-2'-yl)phenoxy)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 RT for 3 h and concentrated in vacuo. The mixture was then purified using reverse phase preparative HPLC to give the 2,2,2-trifluoroacetic acid compound along with 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 (CS0868) (3:1). MS (M+) 852.9.

[0202] Compound CS0858 [ka] Step 1: tert-Butyl (3-oxopropyl)carbamate (17 mg, 0.1 mmol, 1.0 equiv.) was mixed with NaBH(OAc)3 (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 which a solution of 3-(4-(5-(4-methylpiperazin-1-yl)-1H,3′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)propan-1-amine (48 mg, 0.1 mmol, 1.0 equiv.) was added. The reaction was stirred at RT for 24 h 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.

[0203] 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)propanamido)propyl)-c-albamate (64 mg, 0.1 mmol, 1.0 equiv.) and the solution was stirred at RT for 3 h 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]imidazo]-2′-yl)phenoxy)propyl)propanamido)propyl)carbamate.

[0204] 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 RT 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.

[0205] 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 RT for 3 h and 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.

[0206] Compound CS0985 [ka] Step 1: 3,8-bis((tert-butoxycarbonyl)amino)-5-(3-iodopropyl)-6-phenylphenanthridin-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 CH3CN (2 mL) at 40° C. for 6 days. The reaction was concentrated and purified by reverse phase preparative HPLC to give 5-(3-(bis(2-((tert-butoxycarbonyl)amino)ethyl)(methyl)ammonio)propyl)-3,8-bis((tert-butoxycarbonyl)amino)-6-phenylphenanthridin-5-ium iodide.

[0207] 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 CF3CO2H / TIPS (2 mL / 0.2 mL) and stirred at RT for 1 h. The reaction was concentrated and purified by reverse phase preparative HPLC to give the trifluoroacetate salt of 3,8-diamino-5-(3-(bis(2-aminoethyl)-(methyl)ammonio)propyl)-6-phenylphenanthridin-5-ium.

[0208] 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 RT for 3 h and 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.

[0209] Compound CS1065 [ka] 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 CH3CN (5 mL) and the reaction was heated to 50° C. for 3 days. The reaction was then concentrated and used directly in the next step without further purification.

[0210] 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 salt (11.6 mg, 0.01 mmol, 1.0 equiv) was dissolved in TFA / TIPS (2 / 0.2 mL) and stirred at RT for 2 h. The reaction was concentrated and purified by reverse phase preparative HPLC to give the trifluoroacetate salt 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.

[0211] 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 CH3CN (2 mL). The reaction was concentrated in vacuo and purified via 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 complete the reaction and concentrated in vacuo to give the trifluoroacetate salt product of N-(2-aminoethyl)-2-(4-(bis(2-chloro-ethyl)amino)phenoxy)acetamide, which was used in the next step without further purification.

[0212] 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 RT for 3 h and 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)dimethylammonio)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.

[0213] Example 3. Viability PCR experiment with Listeria innocua A turbid overnight culture of Listeria innocua was subcultured into Terrific Broth, grown to early / mid-exponential phase, and concentrated to an OD of ~1.0 (~1 x 108 CFU / mL). 200uL of live or dead (heat killed at 95°C for 15 min, HK) Listeria innocua cells were incubated with the compound CS0775 at the indicated concentrations for 90 min at 37°C. Inactivation buffer was added to the reaction tube and the reaction was incubated at room temperature for 15 min. 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 qPCR signal threshold difference between dead and live cells. Using the described viability PCR workflow, live and non-viable cells are clearly distinguished.

[0214] Example 4. Solvent effects on irreversible cell permeabilization A turbid culture of Pseudomonas aeruginosa was subcultured into LB broth, grown to early / mid-exponential phase, and concentrated to an OD of ~1.0 (~1 × 108 CFU / mL). 200uL of live or dead Pseudomonas aeruginosa cells (heat-killed at 95°C for 15 min, HK) were incubated with 20uM CS0775 at 37°C for 90 min. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 min. Nucleic acids were purified using the Maxwell automated purification workflow and dye-based qPCR analysis was performed using primers specific for Pseudomonas aeruginosa. The results are shown in Figure 3. The size of the PCR amplicon is plotted on the horizontal axis. ΔCt is defined as the difference in qPCR signal threshold between dead and live cells. Increasing the concentration of DMSO in the final reaction increases the efficiency of DNA modification, and therefore allows for the discrimination of non-viable cells from viable cells.

[0215] 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 ~1.0 (~1 × 108 CFU / mL). 200 uL of live or dead bacteria (heat-killed at 95 °C for 15 min, HK) were incubated with 10 uM (Listeria) or 50 uM (Pseudomonas) CS0775 for 90 min at 37 °C. Inactivation buffer was added to the reaction tube, and the reaction was incubated at room temperature for 15 min. Nucleic acids were purified using the Maxwell automated purification workflow and dye-based qPCR analysis was performed using primer sets specific for Pseudomonas aeruginosa or Listeria innocua. The results are shown in Figure 4. PCR amplicon size is plotted on the horizontal axis. Live / dead differential (ΔCt) increases with amplicon length.

[0216] 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), grown to early / mid-exponential phase, and concentrated to an OD of ~1.0 (~1 × 108 CFU / mL). 200 uL of live or killed bacteria (heat-killed at 95 °C for 15 min, HK) were incubated with the indicated concentrations of CS0775 for 90 min at 37 °C. Inactivation buffer was added to the reaction tubes, and reactions were incubated at room temperature for 15 min. Nucleic acids were purified using the Maxwell automated purification workflow, and dye-based qPCR analysis was performed using primer sets specific for the bacterial species described. Results are shown in Figure 5. Optimal concentrations of compounds differ between Gram-negative and Gram-positive bacteria.

[0217] Example 7. Viability PCR assay with 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), grown to early / mid-exponential phase, and concentrated to an OD of ~1.0 (~1 × 108 CFU / mL). 200 uL of live or killed (heat-killed at 95 °C for 15 min, HK) cells were incubated with the indicated concentrations of compounds for 90 min at 37 °C. Inactivation buffer was added to the reaction tubes, and the reactions were incubated at room temperature for 15 min. Nucleic acids were purified using the Maxwell automated purification workflow, and dye-based qPCR analysis was performed using primer sets specific for the bacterial species described. Results are shown in Figure 6. Different classes of compounds can effectively discriminate between live and non-viable cells when used as part of a viability PCR procedure.

[0218] 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 1.0 (approximately 1 x 108 CFU / mL). Serial dilutions were performed and 200uL of live Legionella pneumophila cells at the indicated concentrations were incubated with 10uM CS0775 at 37°C for 90 minutes. Inactivation buffer was added to the reaction tubes and the reactions were 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 set of primers and probes specific for Legionella pneumophila. Results are shown in Figure 7. The assay yielded approximately 10 2 It can detect bacteria at concentrations as low as CFU / mL.

[0219] Example 9. Assays in samples containing live and dead cells Turbid cultures of Legionella pneumophila were subcultured into Legionella cultures, grown to early / mid-exponential phase, and concentrated to an OD of ~1.0 (~1 × 108 CFU / mL). Cells were then diluted to 105 CFU / mL. The indicated proportions of live or dead bacteria (heat-killed at 95 °C for 15 min, HK) were mixed (total volume: 200 uL) and incubated with 10 uM CS0775 for 90 min at 37 °C. Inactivation buffer was added to the reaction tubes and the reactions were incubated at room temperature for 15 min. Nucleic acids were purified using the Maxwell automated purification workflow and probe-based qPCR analysis was performed using a set of primers and probes specific for Legionella pneumophila. The results are shown in Figure 8. The assay is able to differentially detect a small percentage of live cells in a background of mainly dead cells.

[0220] Example 10. Comparison of viability PCR method and conventional culture method Turbid cultures of Legionella pneumophila were subcultured into Legionella broth, grown to early / mid-exponential phase, and concentrated to an OD of 1.0 (approximately 1 x 108 CFU / mL). Serial dilutions were performed and plated on Legionella agar to confirm CFU / mL. In parallel, 200uL of viable Legionella pneumophila cells were incubated with or without 10uM CS0775 for 90 minutes at 37°C. Inactivation buffer was added to the reaction tubes and the reactions were 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 set of primers and probes specific for Legionella pneumophila. A standard curve of Legionella template DNA was amplified simultaneously with the experimental samples to allow quantification. Results are shown in Table 2. vGU: viable genome units. This assay yields quantitative data comparable to traditional culture-based Legionella pneumophila detection techniques. [Table 3]

[0221] Example 11. Viability PCR experiments using adeno-associated virus (AAV) AAV9 reference capsid containing recombinant CMV-GFP plasmid (Vigene Biosciences; Rockville, MD) was diluted to approximately 108 / mL in PBS. 200 uL of live (untouched) or dead (heat inactivated at 75°C for 10 min, HI) AAV9 suspension was incubated with 1 uM CS0775 for 60 min at 37°C. Inactivation buffer was added to the reaction tube and the reaction was incubated at room temperature for 15 min. Nucleic acids were purified using the Maxwell automated purification workflow and dye-based qPCR analysis was performed using primers specific for GFP. The results are shown in Figure 9. Using the described viability PCR workflow, inactivated and heat inactivated viral capsids can be clearly distinguished.

[0222] Example 12. SARS-CoV-2 virus infectivity test Three RT-qPCR assays with different amplicon lengths were designed for use in capsid integrity PCR (Table 3). Regions predicted to have higher secondary structure were selected as amplicons. [Table 4]

[0223] Nasal swab specimens from SARS-CoV-2 infected individuals were diluted 1:100 in PBS. Each dilution was divided into two portions. Samples were treated differently (untreated, 10 min at 70°C, 10 min at 100°C, and 10 min at 70°C in the presence of 0.2% CTAB). One portion was treated with 50uM CS0775 (a viability crosslinker) and the other was mock treated (same volume of DMSO). Samples were vortexed briefly and incubated at 37°C for 60 min. The reactions were then added with Neutralization Solution at 1x concentration, mixed well, and further incubated at ambient temperature for 15 min. This part of the procedure was performed in a BSL2 location.

[0224] Nucleic acid was extracted using the Maxwell® GMO PureFood Authentication kit. Briefly, 10 ul of Protease K and 200 ul of CTAB buffer were added to the virus sample and the mixture was incubated for 30 minutes. This mixture and lysis buffer were added to well #1 of the Maxwell® cartridge and nucleic acid was extracted using the Maxwell® automated particle handler. Nucleic acid was extracted with 80 ul of nuclease-free water. RT-qPCR was performed in 20 μL of amplification reaction consisting of 15 μL of reaction master mix and 5 uL of nucleic acid using the GoTaq® Enviro RT-qPCR system with N1 forward primer (5'-GACCCCAAAATCAGCGAAAT-3'), N1 probe (5'-FAM-ACCCGCAT-ZEN-TACGTTTGGTGGACC-IABkFQ-3'), and N3 reverse primer (5'-TGTAGCACGATTGCAGCATTG-3'). Five μL of nuclease-free water was used as a no-template control (NTC). RT-qPCR reactions were performed using a BioRad CFX96 real-time thermal cycler with the following cycling conditions: reverse transcription at 45° C. for 15 min, initial denaturation at 95° C. for 2 min, and 40 cycles of 95° C. for 3 s and 60° C. for 30 s. The Ct values ​​of each sample and the difference (DCt) between the Ct values ​​of CS0775-treated and untreated samples were calculated. The DCt value was directly proportional to the amount of membrane-compromised virus and thus served as a surrogate for viral infectivity (FIG. 10).

[0225] Example 13. AAV viral infectivity assay Information regarding the infectivity of adeno-associated virus (AAV) can aid in determining dosage in gene therapy applications. AAV does not have a membrane envelope, but has a capsid composed of three capsid proteins VP1, VP2, and VP3. Experiments were performed using AAV reference material (from Vigene Biosciences) during development of the embodiments herein. The AAV contained a plasmid containing a GFP insert. First, we determined whether the multifunctional crosslinker penetrates AAV-8 particles by treating AAV-8 particles with the multifunctional crosslinker CS0775. The addition of 20 uM of the multifunctional crosslinker did not significantly change the Ct value, indicating that the multifunctional crosslinker does not penetrate (Figure 11A). Next, the AAV-8 particles were heat-treated at 75°C for 10 minutes. The 480 bp amplicon within GFP was used for analysis (Figure 11B). The heat-treated AAV-8 samples had a significant right shift in Ct values. This indicates that the viral particles were compromised and that the multifunctional cross-linker was able to cross-link the DNA genetic material and inhibit PCR amplification.

[0226] A second AAV serotype, AAV9, was also tested (Figure 11C). AAV-9 was treated with various concentrations of crosslinker (1, 5, 10, 20 uM, DNase 10 U or DNase + 1 uM multifunctional crosslinker). Three different lengths of amplicons were analyzed. Compared to DNase, the crosslinker was able to inhibit the free DNA signal.

[0227] It should be understood that the above detailed description and the 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.

[0228] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the disclosure, including but not limited to the chemical structures, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use.

Claims

1. A polyfunctional compound or a salt thereof, the polyfunctional compound comprising: (A) RNA binding portion (“RAB portion”), (B) a live cell / dead cell differentiation portion ("LDCD portion"), and (C) the polyfunctional compound or a salt thereof, comprising a nucleic acid modifying moiety ("NAM moiety").

2. The polyfunctional compound has the following structure: A-B-C (wherein A is an RAB moiety, B is an LDCD moiety, and C is a nucleic acid modifying moiety). The polyfunctional compound according to claim 1 or a salt thereof,

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

4. The multifunctional compound or salt thereof according to any one of claims 1 to 4, wherein the RAB moiety comprises a structure of formula (I) or a tautomer or salt thereof: 【Chemistry 1】 (In the formula, R 1 and R 2 are each independently hydrogen, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, aryl, and aryl-C 1 -C 6 alkyl, each of which is optionally substituted with 1 to 3 substituents, or R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl or heteroaryl ring; and R 3 does not exist or C 1 -C 6 It is alkyl.

5. R 1 and R 2 However, each independently, C 1 -C 6 alkyl; R 3 does not exist or C 1 -C 6 The polyfunctional compound of claim 4, wherein the polyfunctional compound is an alkyl group.

6. R 1 , R 2 , and R 3 However, each independently, C 1 -C 6 5. The multifunctional compound of claim 4, wherein the alkyl is selected from the group consisting of aryl, ... and aryl.

7. R 1 and R 2 However, each independently, C 1 -C 6 alkyl (e.g., methyl or ethyl); R 3 The polyfunctional compound according to claim 4 , wherein:

8. R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 5- or 6-membered heterocyclyl or heteroaryl ring having 1, 2 or 3 heteroatoms independently selected from N, O and S.

9. R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form a pyrrolidinyl, piperidinyl, morpholino, piperazinyl, or imidazolyl ring, each of which optionally contains one substituent (e.g., C 1 -C 6 5. The polyfunctional compound of claim 4, wherein the polyfunctional compound is substituted with (alkyl).

10. The group -NR in the moiety of formula (I) 1 R 2 R 3 The multifunctional compound of claim 4, wherein has a formula selected from the following: 【Chemistry 2】

11. 5. The multifunctional compound of claim 4, wherein the RAB moiety has a structure selected from the following: 【Chemistry 3】

12. 12. The multifunctional compound or salt thereof according to any one of claims 1 to 11, 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.

13. The multifunctional compound according to any one of claims 1 to 12, wherein the NAM moiety has a structure selected from the following: 【Chemistry 4】

14. The multifunctional compound or salt thereof according to any one of claims 1 to 13, wherein the LDCD moiety comprises at least one charged moiety.

15. The multifunctional compound of claim 14, wherein the LDCD portion comprises at least one quaternary ammonium group.

16. The polyfunctional compound or salt thereof according to any one of claims 1 to 15, wherein the LDCD moiety comprises at least one poly(ethylene glycol) moiety.

17. 17. The multifunctional compound of claim 16, wherein 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.

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

19. The multifunctional compound or salt thereof according to any one of claims 1 to 13, wherein the LDCD moiety comprises a metabolically cleavable group.

20. The polyfunctional compound according to any one of claims 1 to 19, wherein the polyfunctional compound is selected from the following compounds and any salts thereof: 【Chemistry 5】

21. 1. A method for detecting viable microorganisms or living cells in a sample, the method comprising: (a) contacting a sample with a compound according to any one of claims 1 to 20 or a salt thereof to form a first mixture; (b) contacting the first mixture with a deactivating agent to form a second mixture; and (c) amplifying nucleic acid from the second mixture to produce a detectable signal, the signal indicating the presence of viable microorganisms or living cells in the sample; The method comprising:

22. 22. The method of claim 21, wherein the method does not include a photoactivation step.

23. 23. The method of claim 21 or claim 22, wherein the method does not include a culture step.

24. The method of any one of claims 21 to 23, wherein step (a) comprises contacting the sample with the polyfunctional compound or a salt thereof for between 5 minutes and 180 minutes.

25. 25. The method of claim 24, wherein step (a) comprises contacting the sample with the polyfunctional compound or a salt thereof for between 60 minutes and 120 minutes.

26. The method of any one of claims 21 to 25, wherein step (a) comprises adding to the sample a composition comprising a polyfunctional compound or a salt thereof in a solvent.

27. 27. The method of claim 26, wherein the solvent is dimethylsulfoxide.

28. 28. The method of any one of claims 21 to 27, wherein in step (a), the first mixture comprises a concentration of the multifunctional compound of from 5 to 100 micromolar.

29. The method of any one of claims 21 to 28, wherein the deactivating agent comprises a nucleophile selected from an amine and a thiol.

30. 30. The method of claim 29, wherein the inactivation agent is selected from cysteine, glutathione, dNTPs, guanine, amine-containing buffers, and any mixtures thereof.

31. Step (c) (i) lysing the cells in the second mixture to form a lysed sample; (ii) adding a reverse transcriptase, a DNA replicating enzyme, 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; The method according to any one of claims 21 to 30, comprising:

32. 32. The method of claim 31, further comprising removing contaminants and / or cellular debris from the lysed sample prior to adding the reverse transcriptase, DNA replication enzymes, and amplification reagents.

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

34. 34. The method of claim 33, wherein the amplification reagents comprise forward and reverse primers for a target amplicon in the sample.

35. 22. The method of claim 21 , wherein the detectable signal is a fluorescent signal.

36. The method according to any one of claims 21 to 35, wherein the microorganism is an RNA virus.

37. 37. The method of claim 36, wherein the RNA virus is of the Nidovirales, Picornavirales, or Tymovirales.

38. 38. The method of claim 37, wherein the RNA virus is of the order Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Caliciviridae, Flaviviridae, or Togaviridae.

39. 37. The method of claim 36, wherein the RNA virus is a human pathogen.

40. 40. The method of claim 39, wherein the RNA virus is selected from SARS-CoV-2, HIV-1 rhinovirus, Hepatitis A virus, Yellow Fever virus, West Nile virus, Hepatitis C virus, Dengue virus, and Zika virus.

41. A method for removing nucleic acids from a sample, the method comprising contacting the sample with a compound according to any one of claims 1 to 20.

42. 42. The method of claim 41, wherein the polyfunctional compound is immobilized on a solid support.

43. A system or kit comprising the compound according to any one of claims 1 to 20 or a salt thereof.

44. 44. The system or kit of claim 43, further comprising a reverse transcriptase and a DNA replicating enzyme.

45. 45. The system or kit of claim 44, further comprising one or more amplification reagents.

46. 46. ​​The system or kit of claim 45, further comprising forward and reverse primers for a target nucleic acid sequence of an RNA virus.

47. 47. The system or kit of claim 46, wherein the RNA virus is of the Nidovirales, Picornavirales, or Tymovirales.

48. 48. The method of claim 47, wherein the RNA virus is of the order Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Caliciviridae, Flaviviridae, or Togaviridae.

49. 47. The method of claim 46, wherein the RNA virus is a human pathogen.

50. 50. The method of claim 49, wherein the RNA virus is selected from SARS-CoV-2, HIV-1 rhinovirus, Hepatitis A virus, Yellow Fever virus, West Nile virus, Hepatitis C virus, Dengue virus, and Zika virus.

51. A polyfunctional compound or a salt thereof, the polyfunctional compound comprising: (A) Nucleic acid binding portion (“NAB portion”); (B) Live cell / dead cell differentiation portion (“LDCD portion”); (C) a nucleic acid modification moiety (“NAM moiety”); and (D) an affinity moiety or a conjugation moiety; The polyfunctional compound or a salt thereof.

52. The polyfunctional compound has the following structure: A-B(D)-C where A is an NAB moiety, B is an LDCD moiety, C is a nucleic acid modifying moiety, and D is an affinity or conjugation moiety.

52. The multifunctional compound of claim 51, or a salt thereof, having the formula:

53. 52. The multifunctional compound or salt thereof of claim 51, wherein the multifunctional compound has one or more NAB moieties, one or more LDCD moieties, and / or one or more NAM moieties.

54. 52. The multifunctional compound or salt thereof of claim 51, wherein the NAB is an RNA binding moiety ("RAB moiety").

55. 55. The multifunctional compound of claim 54, wherein the RAB moiety comprises the structure of formula (I) or a tautomer or salt thereof: 【Chemistry 6】 (In the formula, R 1 and R 2 are each independently hydrogen, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, aryl, and aryl-C 1 -C 6 alkyl, each of which is optionally substituted with 1 to 3 substituents, or R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl or heteroaryl ring; and R 3 does not exist or C 1 -C 6 It is an alkyl group.

56. R 1 and R 2 However, each independently, C 1 -C 6 alkyl; R 3 does not exist or C 1 -C 6 56. The multifunctional compound of claim 55, which is alkyl.

57. R 1 , R 2 , and R 3 However, each independently, C 1 -C 6 56. The multifunctional compound of claim 55, wherein the polyfunctional group is selected from alkyl.

58. R 1 and R 2 However, each independently, C 1 -C 6 alkyl (e.g., methyl or ethyl); R 3 The multifunctional compound of claim 55, wherein is absent.

59. R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 5- or 6-membered heterocyclyl or heteroaryl ring having 1, 2 or 3 heteroatoms independently selected from N, O and S.

60. R 3 does not exist, and R 1 and R 2 together with the nitrogen atom to which they are attached form a pyrrolidinyl, piperidinyl, morpholino, piperazinyl, or imidazolyl ring, each of which optionally contains one substituent (e.g., C 1 -C 6 56. The multifunctional compound of claim 55, wherein said compound is substituted with (alkyl).

61. The group -NR in the moiety of formula (I) 1 R 2 R 3 56. The multifunctional compound of claim 55, wherein has a formula selected from the following: 【Chemistry 7】

62. 56. The multifunctional compound of claim 55, wherein the RAB moiety has a structure selected from the following, or a salt thereof: 【Chemistry 8】

63. 52. The multifunctional compound or salt thereof of claim 51, wherein the NAB is a DNA binding moiety ("DAB moiety").

64. 64. The multifunctional compound or salt thereof of claim 63, wherein the DAB moiety is a groove binding moiety, an intercalating moiety, or a mixed mode binding moiety.

65. 64. The multifunctional compound or salt thereof of claim 63, wherein the DAB moiety comprises a bibenzimidazole moiety or a phenylphenanthridium moiety.

66. 64. The multifunctional compound of claim 63, wherein the DAB moiety has a structure selected from the following: or a salt thereof: 【Chemistry 9】

67. 67. The multifunctional compound or salt thereof according to any one of claims 51 to 66, 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.

68. 67. The multifunctional compound of any one of claims 51 to 66, wherein the NAM moiety has a structure selected from the following: 【Chemistry 10】

69. 69. The multifunctional compound or salt thereof according to any one of claims 51 to 68, wherein the LDCD moiety comprises at least one charged moiety.

70. 70. The multifunctional compound of claim 69, wherein the LDCD portion comprises at least one quaternary ammonium group.

71. 71. The multifunctional compound or salt thereof according to any one of claims 51 to 70, wherein the LDCD moiety comprises at least one poly(ethylene glycol) moiety.

72. 72. The multifunctional compound of claim 71, wherein 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.

73. The multifunctional compound according to any one of claims 51 to 72, or a salt thereof, wherein the LDCD moiety comprises one or more of the following groups: 【Chemistry 11】

74. The LDCD portion is one or more alkylene groups (-(CH 2 ) n -), ether group (-O-), thioether group (-S-), ester bond (-C(O)O-), carbamate bond (-OC(O)NH-), sulfonamide bond (-S(O) 2 74. The multifunctional compound or salt thereof of claim 73, further comprising:

75. The multifunctional compound or salt thereof according to any one of claims 51 to 74, wherein the LDCD moiety is a branched LDCD moiety.

76. 76. The multifunctional compound of claim 75, wherein the branched LDCD moiety comprises one of the following branch point functional groups: 【Chemistry 12】

77. 77. The multifunctional compound of claim 76, wherein the branched LDCD moiety comprises: 【Chemistry 13】

78. 78. The multifunctional compound or salt thereof according to any one of claims 51 to 77, wherein the multifunctional compound comprises an affinity moiety.

79. 79. The multifunctional compound or salt thereof of claim 78, wherein the affinity moiety is selected from biotin, a peptide tag, and an epitope.

80. 80. The multifunctional compound of claim 79, comprising the following structure: 【Chemistry 14】

81. 78. The multifunctional compound or salt thereof according to any one of claims 51 to 77, wherein the multifunctional compound comprises a conjugated moiety.

82. 82. The multifunctional compound or salt thereof of claim 81, wherein the conjugated moiety comprises a haloalkane.

83. The haloalkane has the structure -(CH 2 ) n -Y, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and Y is F, Cl, Br or I, or a salt thereof.

84. 84. The multifunctional compound or salt thereof according to claim 83, wherein n is 6 and Y is Cl.

85. A system or kit comprising the compound or salt thereof according to any one of claims 51 to 84.

86. 86. The system or kit of claim 85, further comprising a reverse transcriptase and / or a DNA replication enzyme, and one or more amplification reagents and / or primers.

87. 86. The system or kit of claim 85, further comprising an affinity or conjugation agent.

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