Separation and isolation of nucleic acids using affinity ligands bound to a solid surface

Abstract

Disclosed is a method for isolating and separating target macromolecules, such as DNA (double- or single-stranded), RNA (double- or single-stranded), messenger RNA, or other oligonucleotides or oligonucleosides, from a sample by binding the target macromolecule to a surface-bound affinity ligand. The method can be used in chromatography or any other separation science.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 939,934, filed on November 25, 2019. The entire contents of the foregoing application are incorporated herein by reference.

[0002] The present disclosure relates to a method for separating, isolating, and extracting a target macromolecule such as DNA and RNA from a feed stream, or generally from a sample, using a specifically selected affinity ligand bound to a surface.

Background Art

[0003] When generally used, chromatography is a technique for separating various components of a sample mixture. In a liquid chromatography system, a sample, followed by an eluent, is injected into a chromatography separation column. The separation column contains a packed or matrix medium or material that interacts with the various components of the sample to be separated. The composition of the separation medium depends on the fluid directed through it to effect the desired separation. As the sample and the eluent pass through the separation medium, the various components of the sample move through the separation medium at different rates as a result of differential interactions. These components are separated from the separation medium and appear in the outlet or effluent.

[0004] Various types of vertical and horizontal flow separation columns are known in the art. With the need for high - performance chromatography, horizontal - flow type chromatography columns have been developed. Such horizontal or radial - flow columns are described, for example, in U.S. Patent Nos. 4,627,918 and 4,676,898. In a horizontal or radial - flow type column, the sample and the eluent are introduced via a distributor to the outer periphery or the peripheral wall or the surface of the separation medium or matrix, and the fluid passes through the separation medium horizontally or radially inwardly to a central or collection port and then elutes from the column at different times and different rates.

[0005] Subsequently, chromatography columns and methods have been developed for directly processing crude feed materials to isolate bioactive materials, including cell / fermentation harvests, tissue extracts, and plasma / blood. A large bead chromatography medium is packed into a standard low-pressure chromatography column in which the end plate screen is replaced with a large pore screen (60 - 180 μm pores). The large pores prevent column blockage. Cellular materials, being of large particle size, flow between the beads in the interstices of the inner cavity, while soluble products are captured by functional groups on the beads.

[0006] Traditionally, downstream processing of biological substances derived from cell culture / fermentation harvests has required two main operations: recovery and purification. Recovery involves removing cellular and other particulate materials by centrifugation and / or microfiltration, and a step to reduce the initial volume, generally involving ultrafiltration. Since conventional chromatography media become rapidly fouled by cell debris, a particle-free feed material must be prepared for the purification operation.

[0007] In certain purification processes of (therapeutic) biological preparations (e.g., monoclonal antibodies), the sample / product is produced via a live cell system (mammal, bacterium, moss, algae, plant, etc.), and the product is secreted by the cells into the feed stream or the cells are disrupted and the product is released into the surrounding liquid. However, in all of these production systems, the product is not available as a pure product of a single component, but is a very complex mixture of the desired product and "contaminants" including host cell proteins (HCP) as well as genomic DNA and RNA.

[0008] During purification, HCP and DNA / RNA must be removed from the purified product to levels below those set by regulatory authorities (e.g., the FDA). This purification is usually achieved by anion ion exchange methods. During the filtration and purification of a protein-containing sample, it can be difficult to separate the protein from other cellular components and host proteins without denaturing the protein of interest, and similarly to isolate the target protein. A common method is by precipitation. However, this can lead to the denaturation or degeneration of the protein, which may result in the loss of protein function. Protein refolding often leads to a loss of activity. Fast Protein Liquid Chromatography (FPLC) is a common method used for protein purification. Without the need to denature the protein, high pressure or aggressive pH buffers can be used to purify the protein using only the specific interaction between the buffer and the protein of interest for isolation.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0010] [Non-Patent Document 1] Reddy et al., "Recent developments in sequence selective minor groove DNA effectors", Curr. Med. Chem., 8 (2001), pp. 475-508 [Non-Patent Document 2] Eckel, R. et al., "Identification of Binding Mechanisms in Single Molecule-DNA Complexes", Biophys. J., September 2003, 85(3): 1968-1973 [Non-Patent Document 3] https: / / en.wikipedia.org / wiki / Hoechst_stain [Non-Patent Document 4] ThermoFisher Scientific, Molecular probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, Chapter 8, Nucleic Acid Detection and Analysis, 11th Ed(2010) [Non-Patent Document 5] https: / / www.google.com / url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&ved=2ahUKEwiL5NP478fdAhVqh4sKHffbCFkQFjACegQICBAC&url=http%3A%2F%2Fwww.thermofisher.com%2Fcontent%2Fdam%2FLifeTech%2Fglobal%2Ftechnical-reference-library%2FMolecular%2520Probes%2520Handbook%2Fchapter-pdfs%2FCh-8-Nucleic-Acid-Detection-Analysis.pdf&usg=AOvVaw2Ufpb7SkFbbWTbbzAwdgtm [Non-Patent Document 6] Wiederholt, K. et al., DNA-Tethered Hoechst Groove-Binding Agent: Duplex Stabilization and Fluorescence Characteristics, J. Am. Chem. Soc., 1996, 118, pp. 7055 - 7062 [Non-Patent Document 7] Thompson, M., "Synthesis, photophysical effects, and DNA targeting properties of oxazole yellow-peptide bioconjugates", Bioconjugate Chem. 2006, 17, pp. 507 - 513 [Non-Patent Document 8] Pham, H. H. et al., "Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules", Org. Biomol. Chem. 2015, 13, pp. 3699 - 3710 [Non-Patent Document 9] Fei, X. et al., "Thiazole orange derivatives: synthesis, fluorescence properties, and labeling cancer cells", Bioorg. Med. Chem. 009, 17, pp. 585 - 591 [Non-Patent Document 10] Fei, X. et al., "Solid-phase synthesis and modification of thiazole orange and its derivatives and their spectral properties", J. Comb. Chem. 2007, 9, pp. 943 - 950 [Non-Patent Document 11] Zhang, T. H., He, H. X, Du, J. L., He, Z. J. Yao, S. Molecules, 2018, 23, pp. 2011 - 2024 [Non-Patent Document 12] Gromov, S, P. et al., "Synthesis, Structure, and Properties of Supramolecular Photoswitches Based on Ammonioalkyl Derivatives of Crown Ether Styryl Dyes", J. Org. Chem. 2014, 79, pp. 11416 - 11430

Non - Patent Document 13

Non - Patent Document 14

Non - Patent Document 15

Non - Patent Document 16

Non-Patent Document 17

Summary of the Invention

Problems to be Solved by the Invention

[0011] There is still a need for a system for removing and isolating both double-stranded and single-stranded DNA and RNA from a feed stream.

Means for Solving the Problems

[0012] A method for separating a target macromolecule from a sample, comprising the steps of selecting an affinity ligand that binds to the target macromolecule, binding the affinity ligand to a surface to create a coupled surface-affinity ligand, placing the coupled surface-affinity ligand in a container, introducing a sample containing the target macromolecule into the coupled surface-affinity ligand, and incubating the coupled surface-affinity ligand with the sample for a residence time such that the target macromolecule binds to the affinity ligand, and separating the coupled surface-affinity ligand bound to the target macromolecule from the sample from which the target macromolecule has been removed. Optionally, the method includes the step of collecting an eluate substantially free of the target macromolecule and / or the step of eluting and recovering the target macromolecule from the coupled surface-affinity ligand.

[0013] The target polymer can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, double-stranded messenger RNA, single-stranded messenger RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), virus, protein containing oligonucleotide or oligonucleoside, lipid containing oligonucleotide or oligonucleoside, other oligonucleotides or oligonucleosides, any fragments thereof, or any combination thereof. In certain embodiments, the affinity ligand does not bind to proteins in the sample and / or is methylene blue, a Hoechst dye, a cyanine of benzothiazole-quinoline, or a cyanine of benzoxazole-quinoline. The surface can be a solid surface such as beads, membranes, particles, meshes, polymers, glass, metal, ceramics, silica, polysaccharides, monoliths, or any other material used as a resin in chromatography, including functionalized groups. The container can be a chromatography column, bowl, cylinder, conical vessel, or vat.

[0014] Also disclosed are methods for isolating and extracting DNA from samples containing DNA and other nucleic acids, and methods for isolating and extracting RNA from samples containing RNA and other nucleic acids. These methods include the steps of selecting an affinity ligand that binds to the target DNA or RNA, binding the affinity ligand to a surface to create a coupled surface-affinity ligand, placing the coupled surface-affinity ligand in a container, introducing the sample to the coupled surface-affinity ligand, and incubating the coupled surface-affinity ligand with the sample for a residence time such that the target DNA or RNA binds to the affinity ligand, and separating the coupled surface-affinity ligand bound to the target DNA or RNA from the sample from which the target DNA or RNA has been removed.

Brief Description of the Drawings

[0015]

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Mode for Carrying Out the Invention

[0016] Disclosed herein are methods for capturing and removing target macromolecules from biological feed streams, such as from unclarified (i.e., unfiltered) cell cultures. In certain embodiments, a special capture ligand, which can be a major groove binder, a minor groove binder, or an intercalating ligand (collectively referred to as "affinity ligands"), can be attached to a solid surface, for example, by covalent bonding, and used to irreversibly or reversibly capture target macromolecules, such as DNA, RNA, and lipids and proteins containing oligonucleotides, from complex mixtures such as cell culture feed streams. This method is used in separation science, including but not limited to chromatography, filtration, distillation, and evaporation. Chromatography includes any known method of chromatography, including but not limited to radial flow chromatography, axial chromatography, batch chromatography, adsorption chromatography, expanded bed chromatography, simulated moving bed chromatography, countercurrent chromatography, and high pressure high performance liquid chromatography. Filtration includes any known filtration method in the art, including but not limited to membrane filtration, hollow fiber filtration, and tangential flow / cross flow filtration.

[0017] Disclosed herein are methods for filtering, separating, isolating, removing, and / or purifying nucleic acids using affinity ligands (molecules that exhibit defined interactions with target molecules). The method includes (a) selecting an affinity ligand that binds to a target macromolecule; (b) attaching the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing a sample containing the target macromolecule to the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, such that the target macromolecule binds to the affinity ligand in the container; and (e) separating the coupled surface-affinity ligand, to which the target macromolecule is bound, from the sample from which the target macromolecule has been removed. Optionally, the method further includes (f) collecting an eluate, i.e., the sample from which the target macromolecule has been removed.

[0018] An affinity ligand can be irreversibly immobilized and adhered to a surface such that the affinity ligand can interact and bind with a target macromolecule. The target macromolecule can be a nucleic acid or a fragment thereof. The nucleic acid can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, double-stranded messenger RNA, single-stranded messenger RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), virus, protein containing oligonucleotide or oligonucleoside, lipid containing oligonucleotide or oligonucleoside, other oligonucleotide or oligonucleoside, any fragment thereof, or any combination thereof. The target macromolecule can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, double-stranded messenger RNA, single-stranded messenger RNA, any fragment thereof, or any combination thereof.

[0019] The affinity ligand can be a minor groove binding substance, a major groove binding substance, or an intercalating ligand. The affinity ligand used in each filtration and / or purification process is individually selected such that cross-reactivity (undesirable binding) with molecules other than the target macromolecule is reduced or eliminated. The affinity ligand can be any molecule known to be a minor groove binding substance, a major groove binding substance, or an intercalating ligand, and any further molecule known to be a minor groove binding substance, a major groove binding substance, or an intercalating ligand that also exhibits further selectivity for the target macromolecule.

[0020] In certain embodiments, an affinity ligand can be selected that binds to large-chain DNA and has minimal significant interaction with proteins. An affinity ligand can be selected that binds to double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, double-stranded messenger RNA, single-stranded messenger RNA, any fragment thereof, or any combination thereof, and has minimal significant interaction with other oligonucleotides.

[0021] The surface can be a solid surface that is optionally functionalized to include, for example, ion exchange groups or hydrophobic interaction groups. The surface can have functionalized groups such as spacers ending with epoxy groups, carboxy groups, aldehyde groups, halide groups or amino groups. The spacer refers to a chain of atoms, preferably 1 to 30 or 1 to 20 atoms, attached to the solid surface. The spacer can contain esters, carboxyl groups, or carbon chains (e.g., alkyl). The spacer can contain a polyethylene glycol moiety such as (CH2CH2O)nCH2CH2- (where n = 1 to 10). The spacer is C 1~20 alkylamino, C 1~12 alkylamino, or C 2~12 alkylamino. After the reaction, the spacer enters between the solid surface and the affinity ligand and connects them.

[0022] The solid surface can be beads, membranes, particles, meshes, polymers, glass, metal, ceramic, silica, polysaccharides, monoliths, or any other material used as a resin in chromatography, optionally functionalized as described above. The beads can be agarose beads or amino-agarose beads. The membrane can be an aldehyde membrane such as one made of Sartobind® Aldehyde A4 Sheet. The monolith can be epoxy or ethylenediamine (EDA)-AEX / Activated.

[0023] The surface can be a membrane if the method used is batch chromatography. The surface can be beads if the method used is axial or radial flow chromatography.

[0024] The affinity ligand is bound to the surface, and when a sample or feed stream containing the target macromolecule contacts the affinity ligand, the affinity ligand captures the target macromolecule. The affinity ligand can be bound to the surface by any means known in the art. Examples include, but are not limited to, amide bond formation using carboxylic acids activated with N-hydroxysuccinimide, reaction with aldehydes / Schiff bases, reaction with epoxy groups, click chemistry, and bond formation via Michael addition.

[0025] The bound affinity ligand can be reused or discarded after one use, i.e., it can be a disposable product. For regulatory purposes, disposable products may be preferred to ensure that there is no doubt as to whether any residual contaminants remain after cleaning after each use. Further, cleaning can be very expensive and technically difficult.

[0026] Thus, the affinity ligand selectively binds to the target macromolecule, thereby yielding an eluate substantially free of the target macromolecule. Optionally, if warranted, the target macromolecule is isolated from the remaining sample for recovery and further experimentation and / or processing. The affinity ligand can selectively bind to the target macromolecule but does not bind to proteins. For example, immobilization and isolation of DNA can be performed using amino-agarose beads coupled with a surface-affinity ligand as an intercalating molecule, such as modified methylene blue, or a modified Hoechst dye. After incubation, the DNA binds to the affinity ligand and can be separated from the sample.

[0027] Recovery of DNA from an affinity ligand can be based on ionic interactions between a solid phase such as silica or glass. A binding buffer with a high ionic strength and a pKa below that of the surface silanol groups enables both binding of DNA and washing away of impurities. Subsequently, the DNA is generally eluted using a buffer with a low ionic strength. A disadvantage is that the pH of the cleavage buffer used to remove DNA from the solid phase of the synthetic column is high. The high pH can cause the silica to dissolve and ultimately become an impurity in the DNA.

[0028] After at least a portion or all of the target polymer has been depleted or removed from the feed stream, the eluate can contain less than about 5 wt%, less than about 2 wt%, less than about 1 wt%, less than about 0.5 wt%, less than about 0.2 wt%, less than about 0.1 wt%, less than about 0.05 wt%, less than about 0.01 wt%, less than about 0.005 wt%, or less than about 0.001 wt% of the target polymer.

[0029] In other words, the affinity ligand can bind to at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, or at least about 85 wt% of the target polymer in the sample. The affinity ligand can bind to from about 50 wt% to about 99 wt%, from about 60 wt% to about 98 wt%, or from about 70 wt% to about 98 wt% of the target polymer in the sample.

[0030] An affinity ligand may exhibit minor groove binding or major groove binding to a target macromolecule. Minor groove binding is characterized by selective binding to the narrow minor groove of an AT-rich sequence by van der Waals interactions, hydrogen bonding, and electrostatic interactions. Reddy et al., "Recent developments in sequence selective minor groove DNA effectors", Curr. Med. Chem., 8 (2001), pages 475-508. Major groove binding is characterized by the backbone of the helix ligand and electrostatic interactions with hydrogen bonding. Eckel, R. et al., "Identification of Binding Mechanisms in Single Molecule-DNA Complexes", Biophys. J., September 2003, 85(3): 1968-1973.

[0031] An affinity ligand may exhibit intercalation with a target macromolecule. Intercalation is the reversible insertion of a molecule (or ion) into a material. In certain embodiments, when the target macromolecule is DNA, the affinity ligand interacts with the DNA by intercalating such that a ligand of appropriate size and chemical nature fits between the base pairs of the DNA. Most of these affinity ligands are polycyclic, aromatic, and planar. The affinity ligand may be a dye. The affinity ligand is selected based on the target macromolecule and a selected solid surface. The affinity ligand is not sequence specific. In certain particular embodiments, the affinity ligand is uncharged and does not bind to any protein in the feed stream, if any. If the affinity ligand is charged, the ligand may bind indiscriminately to a protein or other substance. In certain embodiments, when the target macromolecule is DNA, the affinity ligand may be a modified methylene blue, a modified Hoechst dye, a modified thiazole orange, or a modified oxazole yellow that selectively binds to the DNA and does not bind to a protein, if any.

[0032] To isolate, separate, extract, enrich, or purify a target polymer from a sample, reaction, or feed stream, an affinity ligand is modified to be capable of binding to any solid surface (i.e., beads, membranes, particles, meshes, nets, polymers, glass, metal, ceramic, silica, polysaccharides, monoliths, or other solid phases) and capable of binding to one or more target polymers. When the surface is beads, any beads known to be used in chromatography that can also bind to the affinity ligand can be used. The beads can be functionalized glass or agarose, such as glass or agarose conjugated with modified methylene blue, modified Hoechst dye, or modified cyanine of benzothiazole - quinoline or benzoxazole - quinoline, such as modified thiazole orange, modified oxazole yellow. The beads can be glass or agarose having aldehyde groups, carboxylic acid groups, or epoxy groups, such as glass or agarose conjugated with the alkylamino of a modified affinity ligand. When the surface is a membrane, the membrane can bind to modified methylene blue, modified Hoechst dye, or modified cyanine of benzothiazole - quinoline or benzoxazole - quinoline, such as modified thiazole orange, modified oxazole yellow.

[0033] To modify the affinity ligand, first, the optimal attachment points for a linker or spacer group (e.g., attachment points that do not interfere with the site where the ligand recognizes the target polymer) are analyzed or identified, and second, an undesirable and uncontrolled group on the ligand is prevented, and a synthetic strategy for introducing a linker group of appropriate length using standard synthetic techniques known in the art is developed to ensure that one end of the linker group has a suitable functional group for chemically binding to the solid surface.

[0034] An affinity ligand can be any intercalator, minor groove binder, or major groove binder that has been modified to chemically bind to a functionalized surface. The affinity ligand can be selected from acridine, polyimidizole, indole, pyrrole, phenanthridine, benzothiazole-quinoline, or benzoxazole-quinoline cyanines, phenoxazine, phenothiazine, anthraquinone, furanocoumarin, any variants thereof, or any other ligand that binds to the surface and can capture a target macromolecule.

[0035] Acridine structures useful in accordance with the present disclosure include GelGreen (10,10'-(6,22-dioxo-11,14,17-trioxa-7,21-diazaheptacosan-1,27-diyl) bis(3,6-bis(dimethylamino)acridine-10-ium) iodide), acridine orange (N,N,N',N'-tetramethylacridine-3,6-diamine) and its derivatives, amsacrine (synonym: m-AMSA, also known as acridinyl anisidide), and acriflavine (3,6-diamino-10-methylacridine-10-ium chloride) and its derivatives including proflavine (proflavine and diaminoacridine; also referred to as acridine-3,6-diamine), but are not limited thereto.

[0036] Polyimidazole, indole, and pyrrole, which are useful according to the present disclosure, include, but are not limited to, "Hoechst" dyes (i.e., Hoechst 33258, Hoechst 33342 (also known as bisbenzimidazole), and Hoechst 34580) (https: / / en.wikipedia.org / wiki / Hoechst_stain), DAPI (4',6-diamidino-2-phenylindole, a fluorescent stain that binds strongly to adenine-thymine-rich regions of DNA), lexitropsin, netropsin (a polyamide also known as congosidine or sinanomycin), and distamycin (a polyamide antibiotic that acts as a minor groove binding substance and is also known as herpetin, stallymycin), which are known to intercalate into DNA / RNA.

[0037] Lexitropsin is a member of a family of semi-synthetic DNA-binding ligands. Lexitropsin can bind to the minor groove of DNA. Lexitropsin forms complexes with DNA in 1:1 and 2:1 stoichiometries. Lexitropsin has the following structure:

[0038] [Chemical formula]

[0039] It can have, but is not limited to, the following:

[0040] Phenanthridine structures useful according to the present disclosure include phenanthridine and its derivatives, such as ethidium bromide, propidium iodide, propidium monoazide, and GelRed (5,5'-(6,22-dioxo-11,14,17-trioxa-7,21-diazaheptacosan-1,27-diyl) bis(3,8-diamino-6-phenylphenanthridin-5-ium) iodide), etc., but are not limited to these.

[0041] Useful cyanines of benzothiazole - quinoline and benzoxazole - quinoline structures according to the present disclosure include, but are not limited to, dyes of the "Sybr Green" family (Sybr Green I (N',N'-dimethyl - N - [4 - [(E)-(3 - methyl - 1,3 - benzothiazol - 2 - ylidenemethyl]-1 - phenylquinolin - 1 - ium - 2 - yl]-N - propylpropane - 1,3 - diamine), Sybr Green II, Sybr Gold, and Sybr Safe ((Z)-4 - ((3 - methylbenzothiazol - 2(3H)-ylidenemethyl)-1 - propylquinolin - 1 - ium 4 - methylbenzenesulfonate)), dyes of the TOTO(trademark) family and their derivatives, dyes of the YOYO(trademark) family and their derivatives, dyes of the YO - PRO(trademark) family and their derivatives, dyes of the TO - PRO(trademark) family and their derivatives, dyes of the POPO(trademark) family and their derivatives, dyes of the BOBO(trademark) family and their derivatives, dyes of the LOLO(trademark) family and their derivatives, dyes of the JOJO(trademark) family and their derivatives, (ThermoFisher Scientific, Molecular probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, Chapter 8, Nucleic Acid Detection and Analysis, 11th Ed(2010), available below:https: / / www.google.com / url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&ved=2ahUKEwiL5NP478fdAhVqh4sKHffbCFkQFjACegQICBAC&url=http%3A%2F%2Fwww.thermofisher.com%2Fcontent%2Fdam%2FLifeTech%2Fglobal%2Ftechnical-reference-library%2FMolecular%2520Probes%2520Handbook%2Fchapter-pdfs%2FCh-8-Nucleic-Acid-Detection-Analysis.pdf&usg=AOvVa (see w2Ufpb7SkFbbWTbbzAwdgtm), thiazole orange and its derivatives, oxazole yellow and its derivatives, Pico Green and its derivatives

[0042]

Chemical formula

[0043] and dyes of the LightCycler® Green and LightCycler® Red families, but are not limited thereto.

[0044] Phenoxazines useful according to the present disclosure include, but are not limited to, 7-aminoactinomycin D, actinomycin D and its derivatives.

[0045] Phenothiazines useful according to the present disclosure include, but are not limited to, methylene blue (also known as methylthioninium chloride) and its derivatives, including dicarboxymethylene blue-NHS ester (DCMB-SE), and monocarboxymethylene blue (MCMB).

[0046]

Chemical formula

[0047] Anthraquinone structures useful according to the present disclosure include, but are not limited to, anthracyclines and their derivatives, daunorubicin (also known as daunomycin), and doxorubicin.

[0048] Mitoxantrone, losoxantrone (an anthraquinone anthrapyrazole antineoplastic agent and an analogue of mitoxantrone), pixantrone, pirarubicin, and other anthraquinone analogues (e.g., anthraquinone-2-amidophenylcarboxylic acid NHS ester)

[0049] Useful furocoumarin structures according to the present disclosure include psoralen, angelicin, bergamottin ((E)-4-[(3,7-dimethyl-2,6-octadienyl)oxy]-7H-furo[3,2-g][1]benzopyran-7-one) and its derivatives, and amicerin (C 18 H 21 NO5) and its derivatives, but are not limited thereto.

[0050] Other affinity ligands useful according to the present disclosure are those that have mainly ionic interactions with DNA, such as methyl green (C 27 H 35 Cl2N3), and ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), but are not limited thereto.

[0051] The affinity ligand can be any of the above acridine, polyimidazole, indole, pyrrole, phenanthridine, benzothiazole - quinoline or benzoxazole - quinoline cyanines, phenoxazine, phenothiazine, anthraquinone, furocoumarin, or other ligands that bind to the surface and can capture the target macromolecule. The affinity ligand is modified by either a) attaching a linker group to a functional group already present in the compound or dye and then binding that linker group to the surface, or b) chemically modifying the compound or dye to include a linker group and then binding that linker group to the surface, and can be an intercalator, minor groove binding substance, major groove binding substance, or a combination thereof. The linker group can be any reactive linker containing a spacer ending in an epoxy group, carboxy group, aldehyde group, halide group or amino group. The spacer refers to a chain of atoms, preferably 1 to 30, or 1 to 20 atoms, attached to the affinity ligand. The spacer can contain an ester, carboxyl group, or carbon chain. The spacer can contain a polyethylene glycol moiety such as (CH2CH2O) n CH2CH2- (where n = 1 to 10), etc. The spacer is C1~20 Alkylamino, C 1~12 Alkylamino, or C 2~12 It may be alkylamino. The halide may be any halogen or may be F, Cl, Br or I. After the reaction, the spacer enters between the surface and the affinity ligand and connects them. The linker group is C 2~12 Alkylamino, or C 2~8 It may be an alkylamino group such as alkylamino.

[0052] In certain embodiments, the affinity ligand is methylene blue, a Hoechst dye, thiazole orange, Sybr Green, or oxazole yellow that has been modified to include a linker group capable of binding (or tethering) the affinity ligand to the surface. The affinity ligand can be, for example, a Hoechst dye modified to include a linker group of a spacer ending in an epoxy group, a carboxy group, a halide group or an amino group, but is not limited thereto. The affinity ligand can have the left or right side of the compound be C 2~12 Alkylamine, or C 2~8 It may be a Hoechst dye modified with an alkylamine such as alkylamine. The affinity ligand can be, for example, a thiazole orange modified to include a linker group of a spacer ending in an epoxy group, a carboxy group, a halide group or an amino group, but is not limited thereto. The affinity ligand is C 2~12 Alkylamine, or C 2~8 It may be a thiazole orange modified with an alkylamine such as alkylamine. The affinity ligand can be, for example, an oxazole yellow modified to include a linker group of a spacer ending in an epoxy group, a carboxy group, a halide group or an amino group, but is not limited thereto. The affinity ligand is C 2~12 Alkylamine, or C 2~8It may be oxazole yellow modified with an alkylamine such as an alkylamine. The affinity ligand may be a modified methylene blue such as monocarboxymethylene blue. To create monocarboxymethylene blue, a carboxy group is introduced and added to one side of methylene blue so as to act as a linker for attachment to the surface.

[0053] The affinity ligand may be the CDPI3TFP ester disclosed in column 12 of U.S. Patent No. 8,980,855, the entire content of which is incorporated herein by reference:

[0054]

Chemical formula

[0055] In this compound, tetrafluorobenzyl is a reactive carboxy group and reacts, for example, with an amino group on beads to form an amide bond.

[0056] The affinity ligand may be a modified Sybr Green I such as Sybr Green I modified to contain an amino group. One means for synthesizing the modified Sybr Green I is as follows, in part, as disclosed in U.S. Patent No. 5,658,751 and U.S. Patent Application Publication No. 2010 / 0233710, the entire contents of which are incorporated herein by reference:

[0057]

Chemical formula

[0058] Here, Sybr Green I is modified to contain an amino group that binds permanently to the surface by a covalent bond. For example, the amino group binds to beads containing an epoxy group or a carboxy group that is activated by using an NHS ester reagent, and the carboxy group and the amino group are reacted to form an amide group.

[0059] The C3 amine-modified TO(1) or YO(2) utilized in accordance with the present disclosure can be prepared according to the following synthetic scheme:

[0060]

Chemical formula

[0061] 1-(3-aminopropyl)-4-{[3-methyl-2,3-dihydro-1,3-benzoxazol-2-ylidene]methyl}quinolin-1-ium chloride (2) may also be referred to herein as YO-C3. 1-(3-aminopropyl)-4-{[3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]methyl}quinolin-1-ium chloride (1) may also be referred to herein as TO-C3.

[0062] The affinity ligand can be a modified Hoechst dye, which is a compound of the Hoechst bisbenzimidazole family of dyes to which a linker group capable of attaching the affinity ligand (or in this case the Hoechst dye) to the surface is added. The Hoechst dye can be, but is not limited to, Hoechst 33258, Hoechst 33342, or Hoechst 34580, or a mono-Hoechst dye (i.e., containing one benzimidazole ring as opposed to the normal two benzimidazole ring systems). The modification can be on the right side of the molecule, the left side of the molecule, or both. Hoechst 33342 can be modified with a C2-amine group, a C3-amine group, or a C4-amine group on the hydroxybenzaldehyde side (left side). Hoechst 33342 can be modified with a C2-amine group, a C3-amine group, or a C4-amine group on the piperazine side (right side).

[0063] One means for synthesizing the modified Hoechst 33342 is, in part, as disclosed in Wiederholt, K. et al., DNA-Tethered Hoechst Groove-Binding Agent: Duplex Stabilization and Fluorescence Characteristics, J. Am. Chem. Soc., 1996, 118, pp. 7055-7062, as follows:

[0064]

Chemical formula

[0065] Here, Hoechst 33342 is modified to contain an amino group (on the left side of the molecule) that is covalently and persistently bound to the surface. This compound (10), 3-[4-(5-(4-methyl-1-piperazinyl)-(2,5'-bis-1H-benzimidazol-2-yl]-phenoxy-ethylamine may also be referred to herein as L-Hoechst-C2. For example, the amino group is bound to beads containing an epoxy group or a carboxy group that is activated by using an NHS ester reagent, and the carboxy group and the amino group are reacted to form an amide group.

[0066] Another modified Hoechst 33342 utilized in accordance with the present disclosure is, for example, the C3 amine on the hydroxybenzaldehyde side (left side) produced by the following synthetic scheme:

[0067]

Chemical formula

[0068] This compound (17), 3-[4-(5-(4-methyl-1-piperazinyl)-(2,5'-bis-1H-benzimidazol-2-yl]-phenoxy-propylamine may also be referred to herein as L-Hoechst-C3.

[0069] Another modified Hoechst 33342 utilized in accordance with the present disclosure is the C2 amine on the piperazine side (right side) prepared, for example, by the following synthetic scheme:

[0070] [Chemical Formula]

[0071] This compound (12), 2'-(4-ethoxyphenyl)-6-(4-aminoethyl-1-piperazinyl)-2,6'-bis-1H-benzimidazole, may also be referred to herein as R-Hoechst-C2.

[0072] The modified mono-Hoechst, or mono-imidazole Hoechst, can be modified with a C2-amine group, a C2-amine group or a C4-amine group on the hydroxybenzaldehyde side (left side). The modified mono-Hoechst utilized in accordance with the present disclosure is, for example, the C3-amine on the hydroxybenzaldehyde side (left side) prepared according to the following synthetic scheme:

[0073] [Chemical Formula]

[0074] This compound (7), 3-[4-(6-(4-methyl-1-piperazinyl)-1H-benzimidazol-2-yl]-phenoxy-propylamine, may also be referred to herein as Mono-Hoechst-C3.

[0075] When methylene blue is bound to beads (for example, when monocarboxymethylene blue is bound to amino-agarose beads, etc.), since methylene blue is positively charged, it is predicted to bind to DNA and also to proteins such as bovine serum albumin (BSA) (globular protein). However, surprisingly, it was found that methylene blue binds to DNA but not to proteins, so it was found that methylene blue functions well for the removal of DNA in chromatography. Thereby, DNA can be removed and isolated without removing proteins from the feed stream. DNA can be eluted from methylene blue and recovered for further testing and / or processing if necessary.

[0076] When the coupled surface-affinity ligand is one in which modified methylene blue is bound to agarose beads, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 70% to about 95% of the target macromolecule in the sample can bind to the affinity ligand. When the target macromolecule is DNA, the modified methylene blue can bind to about 70% to about 85% or about 70% to about 80% of the DNA in the sample.

[0077] When the coupled surface-affinity ligand is one in which modified Hoechst dye is bound to agarose beads, at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 70% to about 99%, about 80% to about 99%, about 85% to about 98% of the target macromolecule in the sample can bind to the affinity ligand. When the target macromolecule is DNA, the modified Hoechst dye can bind to about 70% to about 99%, about 80% to about 99%, or about 85% to about 98% of the DNA in the sample. When the target macromolecule is DNA, Mono-Hoechst-C coupled to beads or membranes 2~6 , L-Hoechst-C 2~6 or R-Hoechst-C 2~6can bind to about 70% to about 99%, about 80% to about 99%, or about 85% to about 98% of the DNA in the sample. When the target macromolecule is DNA, Mono-Hoechst-C3, L-Hoechst-C3, or R-Hoechst-C3 coupled to the beads can bind to about 70% to about 99%, about 80% to about 99%, or about 85% to about 98% of the DNA in the sample.

[0078] When the coupled surface-affinity ligand is one that binds modified thiazole orange to agarose beads, at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 60% to about 99%, about 70% to about 99%, about 70% to about 95% of the target macromolecule in the sample can bind to the affinity ligand. When the target macromolecule is DNA, modified thiazole orange can bind to about 60% to about 95%, about 70% to about 90%, or about 75% to about 85% of the DNA in the sample. When the target macromolecule is DNA, TO-C coupled to the beads or membrane 2~8 can bind to about 60% to about 95%, about 70% to about 90%, or about 75% to about 85% of the DNA in the sample. When the target macromolecule is DNA, TO-C coupled to the beads 3~6 can bind to about 60% to about 95%, about 70% to about 90%, or about 75% to about 85% of the DNA in the sample.

[0079] When the coupled surface-affinity ligand is one that binds modified oxazole yellow to agarose beads, at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 60% to about 99%, about 70% to about 99%, about 70% to about 95% of the target macromolecule in the sample can bind to the affinity ligand. When the target macromolecule is DNA, modified oxazole yellow can bind to about 60% to about 98%, about 70% to about 95%, or about 80% to about 95% of the DNA in the sample. When the target macromolecule is DNA, YO-C coupled to the beads or membrane 2~8can bind to about 60% to about 98%, about 70% to about 95%, or about 80% to about 95% of the DNA in the sample. When the target macromolecule is DNA, YO-C3 coupled to the beads can bind to about 60% to about 98%, about 70% to about 95%, about 80% to about 95%, or about 85% to about 95% of the DNA in the sample.

[0080] When coupling the affinity ligand to the surface, the density (μg / mL), which is the number of affinity ligands per volume of the solid surface, can be calculated. The greater the density, the more affinity ligands available for binding to the target macromolecule. The dye ligand density can range from about 5 μg / mL to about 50 μg / mL, or from about 6 μg / mL to about 45 μg / mL.

[0081] After preparing the coupled surface-affinity ligand, it is placed in a container for use in a selected separation process. Any container such as a column, vessel, vat, bowl, cylinder, conical vessel, etc., made of any material known to be used in the art, such as glass, plastic, or metal, can be used in accordance with the present disclosure. The container only needs to be selected to function in the separation science process being performed.

[0082] After placing the coupled surface - affinity ligand in a container, the method of the present disclosure involves introducing a sample containing the target macromolecule and one or more nucleic acids and any other combination of other contaminants into the container such that the surface - affinity ligand coupled to the sample comes into contact (e.g., is mixed or combined) and is incubated together with the sample for a residence time, where the target macromolecule binds to the affinity ligand. When a sample or feed stream containing the target macromolecule is incubated with the affinity ligand, the affinity ligand binds to the surface and captures the target macromolecule. The sample and the affinity ligand must be incubated for a residence time, which is the amount of time necessary for the target macromolecule to find and then bind to the affinity ligand, i.e., they must remain in contact. The residence time will be longer when the binding reaction is slow and shorter when the binding reaction between the target macromolecule and the affinity ligand is fast. As is readily understood in the art, the time required for binding to occur is based on the binding kinetics of the coupling and can vary for each coupling. The residence time can be from about 0.1 minute to about 180 minutes, from about 0.1 minute to about 120 minutes, or from about 0.1 minute to about 90 minutes.

[0083] After the residence time, the coupled surface - affinity ligand that has bound to the target macromolecule can be separated from the sample from which the target macromolecule has been removed by any means known in the art. For example, when using chromatography, the solid can be left in the column and the liquid sample or eluate can be allowed to flow out of the column. When using batch chromatography, the sample can be allowed to flow out and the solid can also be removed from the container.

[0084] After this separation, the target macromolecule can be eluted from the coupled surface - affinity ligand by any means known in the art and recovered for further testing and / or processing. This can be referred to as the affinity ligand purification of the oligonucleotide (as opposed to the removal of contaminants).

[0085] After isolation and recovery of the target polymer, quantification (i.e., determination of the amount of the target polymer in the sample) can be carried out by any method known in the art.

[0086] Another embodiment is a method for isolating and recovering a target polymer from a sample containing the target polymer and other contaminants, nucleic acids, oligonucleosides, or combinations thereof. The method includes: (a) selecting an affinity ligand that binds to the target polymer; (b) binding the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing a sample containing the target polymer into the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time such that the target polymer binds to the affinity ligand in the container; and (e) separating the remaining sample from the coupled surface-affinity ligand (i.e., the solid) to which the target polymer is coupled.

[0087] After this separation, the target polymer can be eluted from the coupled surface-affinity ligand by any means known in the art and recovered for further testing and / or processing. This can be referred to as affinity ligand purification of the oligonucleotide (as opposed to removal of contaminants).

[0088] A method for isolating and extracting DNA from a sample containing DNA and other nucleic acids or oligonucleotides, comprising: (a) selecting an affinity ligand that binds to DNA; (b) binding the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing the sample to the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, such that the DNA binds to the affinity ligand; and (e) separating the coupled surface-affinity ligand bound to the DNA from the sample from which the DNA has been extracted. The DNA can be single-stranded DNA or double-stranded DNA. The method for isolating and extracting DNA from the sample can further include eluting and recovering the DNA from the coupled surface-affinity ligand.

[0089] A method for isolating and extracting RNA from a sample containing RNA and other nucleic acids or oligonucleotides, comprising: (a) selecting an affinity ligand that binds to RNA; (b) binding the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing the sample to the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, such that the RNA binds to the affinity ligand; and (e) separating the coupled surface-affinity ligand bound to the RNA from the sample from which the RNA has been extracted. The RNA can be messenger RNA. The RNA can be single-stranded RNA, double-stranded RNA, or messenger RNA. The method for isolating and extracting RNA from the sample can further include eluting and recovering the RNA from the coupled surface-affinity ligand.

[0090] Another embodiment is a method for isolating and extracting double-stranded DNA from a sample containing double-stranded DNA and other nucleic acids, for example, a sample containing both single-stranded DNA and double-stranded DNA, by selecting an affinity ligand that preferentially binds to double-stranded DNA. The method includes: (a) selecting an affinity ligand that binds to double-stranded DNA; (b) binding the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing a sample containing double-stranded DNA into the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, such that the double-stranded DNA binds to the affinity ligand in the container; and (e) separating the remaining sample from the coupled surface-affinity ligand coupled to the double-stranded DNA by any means known in the art.

[0091] Another embodiment is a method for isolating and extracting double-stranded RNA from a sample containing double-stranded RNA and other nucleic acids, for example, a sample containing both single-stranded RNA and double-stranded RNA, by selecting an affinity ligand that preferentially binds to double-stranded RNA. The method includes: (a) selecting an affinity ligand that binds to double-stranded RNA; (b) binding the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing a sample containing double-stranded RNA into the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, such that the double-stranded RNA binds to the affinity ligand in the container; and (e) separating the remaining sample from the coupled surface-affinity ligand coupled to the double-stranded RNA by any means known in the art.

[0092] Another embodiment is a method of isolating and extracting single-stranded DNA from a sample containing single-stranded DNA and other nucleic acids, for example, a sample containing both single-stranded DNA and double-stranded DNA, by selecting an affinity ligand that preferentially binds to single-stranded DNA but not to double-stranded DNA. The method includes: (a) selecting an affinity ligand that binds to single-stranded DNA; (b) binding the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing a sample containing single-stranded DNA to the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, such that the single-stranded DNA binds to the affinity ligand in the container; and (e) separating the remaining sample from the coupled surface-affinity ligand to which the single-stranded DNA is coupled by any means known in the art.

[0093] Another embodiment is a method of isolating and extracting single-stranded RNA from a sample containing single-stranded RNA and other nucleic acids, for example, a sample containing both single-stranded RNA and double-stranded RNA, by selecting an affinity ligand that preferentially binds to single-stranded RNA but not to double-stranded RNA. The method includes: (a) selecting an affinity ligand that binds to single-stranded RNA; (b) binding the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing a sample containing single-stranded RNA to the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, such that the single-stranded RNA binds to the affinity ligand in the container; and (e) separating the remaining sample from the coupled surface-affinity ligand to which the single-stranded RNA is coupled by any means known in the art.

[0094] Another embodiment is a method of isolating and extracting messenger RNA (double-stranded and / or single-stranded) by selecting an affinity ligand that preferentially binds to messenger RNA but does not bind to other nucleic acids in a sample containing other types of RNA (double-stranded and / or single-stranded) and / or DNA (double-stranded and / or single-stranded) and / or other nucleic acids. The method includes: (a) selecting an affinity ligand that binds to messenger RNA; (b) binding the affinity ligand to a surface to create a coupled surface-affinity ligand; (c) placing the coupled surface-affinity ligand in a container; (d) introducing a sample containing messenger RNA to the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand together with the sample for a residence time, such that the messenger RNA in the container binds to the affinity ligand; and (e) separating the remaining sample from the coupled surface-affinity ligand to which the messenger RNA is coupled by any means known in the art.

[0095] The terms used in connection with these embodiments (methods of use) have the same meanings and definitions as those described above.

[0096] The features and advantages of the present invention are shown in more detail by the following examples. The following examples are presented for illustrative purposes and are in no way to be construed as limiting the present invention.

Examples

[0097] (Example 1) Agarose beads coupled with methylene blue were synthesized and used in radial flow chromatography. When a sample containing a mixture of DNA and protein was placed in a chromatography column and allowed to stand for the required residence time before flowing, the agarose beads coupled with methylene blue bound to approximately 80% of the DNA in the sample. Whether with a small amount of material (100 μL) or on a 1 mL scale-up of the resin, the binding interaction was clearly shown by FPLC analysis. At the same time, no non-specific binding of DNA to the negative control was observed. The saturation level of DNA using this newly designed matrix has not yet been determined. However, it was possible to observe a tendency for the percentage of the DNA binding level to decrease when using a DNA concentration > 310 mg / mL. For very small DNA fragments, a decrease in the DNA binding percentage was also caused. When attempts were made to exclude the smallest DNA fragments by ultrafiltration (exclusion of less than 10 kDa), an improvement was detected.

[0098] Next, agarose beads coupled with monocarboxymethylene blue were incubated with a 1 mg / mL BSA solution to determine whether the intercalating dye interacts with the protein. No protein binding could be detected using either methylene blue agarose or the negative control.

[0099] In summary, agarose resin coupled with methylene blue that can bind to large-chain DNA but does not interact with proteins was synthesized.

[0100] Figures 1A and 1B show a comparison of DNA (<50 bp in size) "ultrafiltered" with untreated and two concentrations of MCMB bead-added agarose gels (30 μg of MCMB per 1 mL of gel and 65 μg of MCMB per 1 mL of gel, MCMB = monocarboxymethylene blue): Figure 1A = % of DNA bound to the gel out of the total DNA added, Figure 1B = the capacity of the gel to bind DNA (μg of DNA per 1 mL of gel). Black = MCMB-agarose; gray = control (no MCMB present on the gel).

[0101] Figures 2A and 2B are comparisons of DNA binding and the capacity of the gel to bind DNA using DNAs of different sizes: Figure 2A = % of the total DNA added that bound to the gel, Figure 2B = capacity of the gel to bind DNA (μg of DNA per mL of gel). White = native agarose beads without amino groups; black = amino-functionalized agarose beads; gray = DNA, either <50 bp or <2000 bp in concentration. The given concentrations (64.8 and 69) are μg of MCMB per mL of gel.

[0102] Figures 3A - 3D show the results of the control: absorbance and conductivity at 260 nm of different concentrations (each 50 μL) of DNA passing through a 1 mL FPLC column packed with native agarose beads. From the graph, it is shown that for four different DNA concentrations (3A = 3.3 mg / mL, 3B = 1.5 mg / mL, 3C = 0.33 mg / mL, 3D = 0.15 mg / mL), no binding was observed and no linearity with concentration was observed (see the height of the peaks on the left scale).

[0103] Figure 4 is a graph regarding the loading of DNA (2.5 mg / mL in TRIS, each 50 μL) onto a 1 mL FPLC column packed with amino-agarose beads (light blue) or native agarose beads (light orange, both on the left scale, the Gaussian curve is the absorbance at 260 nm). The upper "horizontal" curve is the conductivity (right scale).

[0104] Figure 5 shows the results regarding the loading of DNA (2.5 mg / mL in TRIS, each 50 μL) onto a 1 mL FPLC column with MCMB agarose beads. The first loading (black solid line) shows a significant decrease in the DNA passing through the column, and the second loading (black dashed line) is higher due to the saturated column.

[0105] Table 1 (Table 1) shows the amount of DNA bound to agarose beads coupled with methylene blue. NK1 = amino agarose beads, 30% DNA binding (presumably ionic interaction), NK2 = native agarose beads, essentially no DNA binding. MCMB agarose beads, 88% of the DNA in the sample bound in the first loading, and 50% bound in the second loading (presumably due to saturation).

[0106]

Table 1

[0107] Table 2 (Table 2) shows the BSA protein bound to agarose beads coupled with methylene blue. 3.9% bound to NK1 amino-agarose, 5.5% bound to NK2 native-agarose, essentially zero BSA bound to MCMB-agarose, and 11% BSA bound to MCMB-agarose pre-bound with 42 micrograms of DNA. There seems to be no interaction between the BSA protein and the ligand and no partial ionic bond with either the cationic amino group or anionic DNA. The 5.5% native agarose binding of BSA is thought to be due to a small amount of non-specific interaction with the resin.

[0108]

Table 2

[0109] (Example 2) Synthesis of TO-I Thiazole orange-propyl iodide (TO-I) was synthesized as follows:

[0110]

Chemical formula

[0111] S-Methyl-benzothiazole-tosylate 1.84 (5.0 mmol) was mixed with repidine iodide 2.20 mg (5.0 mmol), and then the mixture was dissolved in 10 ml of EtOH. 0.5 ml of Et3N was added, and the mixture immediately turned red and then became a dark brownish red. The solution was stirred at 60 °C for 60 minutes and then left at room temperature for 12 hours. The resulting fine precipitate was vacuum filtered under vacuum and dried. A total of 1.95 g of pure product was obtained → yield 67% UV (MeOH): λmax 506 nm HPLC: Nucleosil 100, C18, 5 μm, MeCN / H2O (98:2), R t for 18.8 minutes MALDI: m / z: 459.8 (C 21 H 20 IN2S + ) 1 1H-NMR (DMSO-d6): 2.35 (m, 2H), 3.33 (s, 3H), 3.95 (m, 2H), 4.55 (m, 2H), 6.79 (s, 1H), 7.20 (d, 1H), 7.34 (tr, 1H), 7.53 (tr, 1H), 7.67 (2d, 2H), 7.92 (tr, 1H), 7.96 (d, 1H), 7.90 (tr, 1H), 8.02 (d, 1H), 8.53 (d, 1H), 8.71 (d, 1H). 13 13C-NMR (DMSO-d6): 2.3, 32.5, 33.9, 54.4, 88.2, 107.7, 112.9, 117.6, 122.7, 123.8, 124.0, 124.4, 125.8, 126.6, 128.0, 133.1, 136.8, 140.2, 144.1, 148.2, 159.9.

[0112] TO-I was bound to agarose beads and then used to bind DNA.

[0113] (Example 3) Synthesis of TO-C3 and YO-C3 Thiazole Orange was modified by introducing a reactive linker, for example, a linker containing an amino group. After attempts to synthesize TO-C3 by a process that was previously thought to be correct failed, TO-C3 was synthesized as follows:

[0114] TO- and YO-C3-amines (1) and (2)

[0115]

Chemical formula

[0116] N-Boc-1-(3-aminopropyl) rhodanine bromide (3)

[0117]

Chemical formula

[0118] 5.8 g (24.5 mmol) of N-Boc-3-bromopropylamine and 2.3 g (16.1 mmol) of rhodanine were uniformly dissolved at 50 - 60 °C for 16 hours without using any solvent. The remaining reaction mixture was extracted several times with ethyl acetate until a solid precipitate was observed. This solid was filtered under vacuum and dried. 4.8 g of N-Boc-rhodanine bromide (3) was isolated (yield 78%).

[0119] N-Boc-TO-C3-amine (9)

[0120]

Chemical formula

[0121] N-Boc-Repidinium (3) 400 mg (1.09 mmol) and benzothiazole tosylate (4) (Thompson, M., "Synthesis, photophysical effects, and DNA targeting properties of oxazole yellow-peptide bioconjugates", Bioconjugate Chem. 2006, 17, 507-513) 1.12 g (3.05 mmol) were dissolved in 50 ml of CH2Cl2 at room temperature (rt.). 2.0 ml (14 mmol) of Et3N was added dropwise at room temperature. A deep red color appeared. After 60 minutes, the completion of the reaction was detected by TLC. The solvent was removed under vacuum (i.vac.), and the residue was purified by sc-chromatography (CHCl3 / MeOH (100 / 0 → 100 / 5)). R f : 0.7 (CHCl3 / MeOH / aqu. NH3: 70 / 28 / 2) MALDI: m / z: 348,2 (C 21 H 21 N3S + : M-CO2tBu) 340 mg of N-Boc protected 10 was isolated (yield 59%)

[0122] 1-(3-Aminopropyl)-4-{[3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]methyl}quinolin-1-ium chloride (TO-C3) (1)

[0123]

Chemical formula

[0124] 400 mg (0.76 mmol) of N-Boc protected 9 was dissolved in 20 ml of abs. MeOH. The clear solution was acidified with HCl / MeOH and stirred at room temperature for 16 hours. The solvent was removed under vacuum. The residue was extracted several times with ethyl acetate, filtered under vacuum, and dried. 250 mg of TO-C3 (1) was isolated (yield 86%). Rf : 0.45 (CHCl3 / MeOH / aq. NH3: 70 / 28 / 2) UV (MeOH): λ max 506 nm HPLC: Nucleosil 100, C18, 5 μm, MeCN / H2O (98:2), R t 11.4 minutes MALDI: m / z: 348.2 (C 21 H 22 N3S + ) 1 1H-NMR (dmso-d6): 2.14 (quintet, J = 7.2 Hz, 2H), 2.87 (t, J = 7.1 Hz, 2H), 4.03 (s, 3H), 4.73 (t, J = 7.3 Hz, 2H), 6.95 (s, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.43 (t, J = 7.3 Hz, 1H), 7.62 (t, J = 7.1 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.99 (t, J = 8.2 Hz, 1H), 8.06 (d, J = 7.2 Hz, 1H), 8.21 (d, J = 8.7 Hz, 1H), 8.74 (d, J = 6.8 Hz, 1H), 8.81 (d, J = 8.1 Hz, 1H). 13 13C-NMR (dmso-d6): 27.0, 33.9, 36.0, 51.2, 88.3, 107.9, 113.1, 118.1, 122.9, 123.9, 124.3, 124.6, 125.9, 126.8, 128.2, 133.3, 137.0, 140.5, 144.2, 148.6, 160.4.

[0125] 1-(3-Aminopropyl)-4-{[3-methyl-2,3-dihydro-1,3-benzoxazol-2-ylidene]methyl}quinolin-1-ium chloride (YO-C3)(2)

[0126]

Chemical Structure

[0127] 10400 mg (0.76 mmol) of N-Boc protection was dissolved in 20 ml of absolute MeOH. The clear solution was acidified with HCl / MeOH and stirred at room temperature for 16 hours. The solvent was removed under vacuum. The residue was extracted several times with ethyl acetate, filtered under vacuum and dried. 250 mg of TO-C3(2) was isolated (yield 86%). R f : 0.45 (CHCl3 / MeOH / aqu. NH3: 70 / 28 / 2) UV (MeOH): λ max 580 nm HPLC: Nucleosil 100, C18, 5 μm, MeCN / H2O (98:2), R t 10.7 minutes MALDI: m / z: 332.5 (C 21 H 22 N3O + ) 1 1H-NMR (dmso-d6): 2.18 (quintet, J = 7.3 Hz, 2H), 2.91 (sextet, J = 6.0 Hz, 2H), 3.87 (s, 3H), 4.74 (tr, J = 7.3 Hz, 2H), 6.30 (s, 1H), 7.39 (tr, J = 7.8 Hz, 1H), 7.48 (tr, J = 7.6 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.72 (tr, J = 7.6 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 7.2 Hz, 1H), 7.97 (tr, J = 7.2 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.62 (d, J = 7.2 Hz, 1H), 8.78 (d, J = 8.4 Hz, 1H). 13C-NMR (dmso-d6): 26.8, 30.5, 35.9, 50.9, 74.2, 109.1, 110.7, 110.9, 117.9, 123.4, 124.3, 125.9, 126.2, 126.5, 131.3, 133.3, 137.1, 143.4, 146.1, 150.0, 161.5.

[0128] (Example 4) Comparative synthesis scheme with the prior art In the prior art, the authors thought that TO-C3 and YO-C3 could be prepared according to the following synthesis scheme. Regarding thiazole orange amine 1 (TO-amine 1), in one report, 1 H- and 13The chemical shifts of the signals in the 13C-NMR spectrum were collected without assignment (Pham, H. H. et al., "Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules", Org. Biomol. Chem. 2015, 13, 3699-3710). On the other hand, incorrect data regarding this compound were published in other reports (Brenner, S. et al., "Fluorescent molecular motors", PCT International Application 2014, WO 2014051521, A1 20140403; Fei, X. et al., "Thiazole orange derivatives: synthesis, fluorescence properties, and labeling cancer cells", Bioorg. Med. Chem. 2009, 17, 585-591; Fei, X. et al., "Solid-phase synthesis and modification of thiazole orange and its derivatives and their spectral properties", J. Comb. Chem. 2007, 9, 943-950). When the experiment was carried out, amidine compounds 7 and 8 were produced instead of the desired TO-C3 and YO-C3. In this experiment, S-methyl-benzothiazole tosylate (4) was in excess and reacted with the right and left sides of the repidinium bromide.

[0129] 1-(3-{[-3-Methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]amino}propyl)-4-{[3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]methyl}quinolin-1-ium bromide (TO-C3-amidine) (7)

[0130] [Chemical formula]

[0131] S-Methyl-benzothiazole tosylate (4) (Zhang, T. H.; He, H. X; Du, J. L.; He, Z. J. Yao, S. Molecules, 2018, 23, pp. 2011 - 2024) 1.26 g (3.6 mmol) and repidinium bromide (10) (Gromov, S, P. et al., "Synthesis, Structure, and Properties of Supramolecular Photoswitches Based on Ammonioalkyl Derivatives of Crown Ether Styryl Dyes", J. Org. Chem. 2014, 79, pp. 11416 - 11430) 980 mg (3.5 mmol) were suspended in 20 ml of CH2Cl2. 2.0 ml (14 mmol) of Et3N was added dropwise. The deep red reaction mixture was stirred for an additional 60 minutes. The completion of the reaction was detected by TLC. The reaction mixture was extracted with saturated NH4Cl and aqueous Na2CO3 solutions. The organic phase was separated, dried (Na2SO4), filtered, and the solvent was evaporated under vacuum. The residue was purified by crystallization (ethanol). 750 mg of TO-C3-amidine 7 was isolated (yield 43%). Rf: (CHCl3 / MeOH NH3 : 85 / 15) UV (MeOH): λ max 506 nm HPLC: Nucleosil 100, C18, 5 μm, MeCN / H2O (98:2), R t 13.8 minutes MALDI: m / z: 495.4 (C 29 H 27 N4S2 + ) 11H-NMR (DMSO-d6): 2.24 (quintet, J = 5.9 Hz, 2H), 3.16 (s, 3H), 3.17 (m, 2H), 4.00 (s, 3H), 4.76 (t, J = 6.7 Hz, 2H), 6.86 (t, J = 7.5 Hz, 1H), 6.87 (s, 1H), 6.99 (d, J = 7.9 Hz, 1H), 7.10 (dt, J = 1.0, 7.3 Hz, 1H), 7.23 (d, J = 7.1 Hz, 1H), 7.41 (t, J = 7.5 Hz), 7.46 (d, J = 6.9 Hz, 1H), 7.61 (dt, J = 7.3 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.99 (t, J = 8.1 Hz, 1H), 8.22 (d, J = 8.7 Hz, 1H), 8.31 (s, 1H), 8.59 (d, J = 7.2 Hz, 1H), 8.77 (d, J = 8.9 Hz, 1H). 13 13C-NMR (DMSO-d6): 29.6, 33.7, 50.7, 53.1, 79.2, 88.0, 107.7, 109.0, 112.9, 118.3, 120.6, 121.2, 122.3, 122.8, 123.9, 124.3, 124.4, 125.7, 126.3, 126.7, 128.1, 133.1, 137.2, 140.4, 140.6, 144.6, 148.5, 154.7, 159.9.

[0132] 1-(3-{[-3-Methyl-2,3-dihydro-1,3-benzoxazol-2-ylidene]amino}propyl)-4-{[3-methyl-2,3-dihydro-1,3-benzoxazol-2-ylidene]methyl}quinolin-1-ium bromide (YO-C3-amidine) (8)

[0133]

Chemical formula

[0134] S-Methyl-benzooxazole tosylate (5) (Gromov, S, P. et al., "Synthesis, Structure, and Properties of Supramolecular Photoswitches Based on Ammonioalkyl Derivatives of Crown Ether Styryl Dyes", J. Org. Chem. 2014, 79, pp. 11416 - 11430) 1.35 g (3.8 mmol) and repidinium bromide (10) 980 mg (3.5 mmol) were suspended in 20 ml of CH2Cl2. 2.0 ml (14 mmol) of Et3N was added dropwise. The deep red reaction mixture was stirred for an additional 60 minutes. The completion of the reaction was detected by TLC. The reaction mixture was extracted with saturated NH4Cl and aqueous Na2CO3 solutions. The organic phase was separated, dried (Na2SO4), filtered, and the solvent was evaporated under vacuum. 750 mg was isolated (yield 36%) R f :(CHCl3 / MeOH NH3 :85 / 15) UV (MeOH): λ max 580 nm HPLC: Nucleosil 100, C18, 5 μm, MeCN / H2O (98:2), R t 10.7 minutes MALDI: m / z: 331.2 (C 21 H 21 N3O + : M-(N-Me-benzooxazole)) 11H-NMR (dmso-d6): 2.13 (quintet, J = 6.0 Hz, 2H), 3.13 (s, 3H), 3.41 (t, J = 6.0 Hz, 2H), 3.82 (s, 3H), 4.70 (t, J = 6.0 Hz, 2H), 6.82 (m, J = 6.0 Hz, 1H), 6.96 (m, J = 6.0 Hz, 1H), 7.00 (p, J = 6.0 Hz, 1H), 7.17 (p, J = 6.0 Hz, 1H), 7.33 (t, J = 6.0 Hz, 1H), 7.43 (t, J = 6.0 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.67 (t, J = 6.0 Hz, 1H), 7.67 (d, J = 6.0 Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H), 7.92 (t, J = 6.0 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H), 8.33 (s, 1H), 8.50 (d, J = 7.2 Hz, 1H), 8.71 (d, J = 8.7 Hz, 1H). 13 13C-NMR (DMSO-d6): 28.3, 29.6, 30.5, 42.2, 52.4, 73.6, 79.2, 107.4 (br), 108.6, 110.5, 110.7, 118.0, 120.3 (br), 123.3, 123.6 (br), 125.8, 126.3, 126.0, 126.3, 131.1, 132.0, 133.1 (br), 137.2, 143.8, 143.8, 145.9, 149.7.3, 161.2.

[0135] (Example 5) Synthesis of 3-[4-(6-(4-methyl-1-piperazinyl)-1H-benzimidazol-2-yl]-phenoxy-propylamine (Mono-Hoechst-C3)

[0136]

Chemical formula

[0137] N-Boc-(4-Formylphenoxy)propylamine (4) (Liu, Y. et al., "A 'Double-Locked' and enzyme-activated molecular probe for accurate bioimaging and hepatopathy differentiation", Chemical Science 2019, 10(47), pp. 10931-10936.)

[0138] [Chemical formula]

[0139] 4-Hydroxyaldehyde 2960 mg (11.7 mmol), K2CO3 2.52 g (8 mmol) and N-Boc-3-bromopropylamine 1 2.36 g (10.0 mmol) were heated in 3 ml of dry DMF for 16 hours. The reaction mixture was diluted with 100 ml of ethyl acetate and extracted 2 - 3 times with saturated NaCl solution. The organic phase was separated, dried (Na2SO4), filtered and evaporated under vacuum. 2.47 g of N-Boc protected aldehyde 4 was isolated (yield 88%). R f : about 0.5 (CHCl3 / ethyl acetate: 88 / 12) C 15 H 21 NO4(279.3) MS-ESI: 280 (M+1), 265 (M-CH3), 223 (M-tBu),

[0140] N-Boc-3-(4-(6-(4-Methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-phenoxy)propyl-1-amine (6) (Ranjan, N. et al., "Selective Inhibition of Escherichia coli RNA and DNA Topoisomerase I by Hoechst 33258 Derived Mono-and Bisbenzimidazoles", J. Med. Chem. 2017, 60, pp. 4904-4922.)

[0141]

Chem.

[0142] 385 g (2.0 mmol) of Na2S2O5 was dissolved in 2 ml of H2O, and 1.06 g (3.8 mmol) of N-Boc protected aldehyde 4 in 5 ml of EtOH was added to this aqueous solution. A yellow to gray precipitate was observed. A solution of 620 mg (3 mmol) of piperazinyl diamine 5 in 50 ml of EtOH was added to the aldehyde suspension. The orange to brown suspension was heated at 60 °C for 1 - 2 hours. Completion of the reaction was detected by TLC. R f : about (CHCl3 / MeOH / aqu. NH3: 80 / 16 / 4) UV: λ max 326 nm, λ max 272 nm. HPLC: Luna 3μ, phenyl - hexyl, MeCN / H2O (98:2), R t 11.8 minutes C 26 H 35 N5O3 (465.6) MS - MALDI: 466.7 (M + 1), 443 (M - 43 (NC2H5))

[0143] 3-(4-(6-(4 - methylpiperazin - 1 - yl)-1H - benzimidazol - 2 - yl)-phenoxy)propyl - 1 - amine (7)

[0144]

Chem.

[0145] 930 mg (2 mmol) of N - Boc protected benzimidazole 6 was dissolved in 10 ml of MeOH. This solution was acidified with HCL / MeOH. After 24 hours at room temperature, complete deprotection was detected by TLC. Mono - Hoechst - C37 was crystallized from the reaction mixture. 400 mg was isolated by filtration (yield 55%). UV: λ max 326 nm, λ max 272 nm HPLC: Luna 3μ, phenyl - hexyl, MeCN / H2O (98:2), R t 3.1 minutes C 21 H 27 N5O (365.5) MS - MALDI: 366.1 (M + 1), 309 (M - 57(NC3H7)) 1 1H - NMR (dmso - d6): 2.09 (quintet, J = Hz, 2H), 2.30 (s, 3H), 2.60 (m, 4H), 2.95 (tr, J = Hz, 2H), 3.16 (m, 4H), 4.15 (tr, J = Hz, 2H), 6.91 (m, 2H), 7.07 (d, J = Hz, 2H), 7.42 (m, J = Hz, 1H), 8.11 (d, J = Hz, 2H). 13 13C - NMR (DMSO - d6):

[0146] (Example 6) Synthesis of 2'-(4 - ethoxyphenyl)-6-(4 - aminoethyl - 1 - piperazinyl)-2,6'-bis - 1H - benzimidazole (R - Hoechst - C2)

[0147]

Chemical formula

[0148] 5 - [4-(2 - N - Boc - aminoethyl)piperazinyl)] - 2 - nitroaniline (8)

[0149]

Chemical formula

[0150] 2.0 g (11.6 mmol) of 5-chloro-2-nitroaniline, 2.0 g (14.4 mmol) of K2CO3 and 4.0 g (17.5 mmol) of 1-(2-N-Boc-aminoethyl)piperazine were suspended in 2.0 ml of dry DMF. The suspension was stirred at 140 - 150 °C for 24 hours. The cooled reaction mixture was dissolved in 100 ml of ethyl acetate and extracted three times with saturated NaCl solution. The organic phase was separated, dried (Na2SO4), filtered and evaporated under vacuum. The residue was purified by sc-chromatography (MeOH / CHCl3: 1 / 9). 4.12 g of N-Boc protected 2-nitroaniline 8 was isolated (yield 97%). HPLC: Luna 3μ, phenyl-hexyl, MeCN / H2O (98:2), R t 15.3 minutes C 17 H 27 N5O4(365.2) MS-MALDI: 366.1 (M), 350.2 (M-Me), 310 (M-C4H8)

[0151] (N-Boc-aminoethyl)-Hoechst 33342(11)

[0152]

Chemical Structure

[0153] 3.0 g (8.2 mmol) of N-Boc protected 2-nitroaniline 8 was dissolved in 160 ml of ethanol and 0.7 g of 10% Pd / C was added. The mixture was stirred under a hydrogen atmosphere for 5 - 6 hours. The complete completion of the reaction was observed by TLC. After filtration of the catalyst (celite), the solution of N-Boc protected diamine 10 was used immediately without any further purification.

[0154] Meanwhile, a solution of 3.2 g (12 mmol) of Hoechst aldehyde 9 (Nimesh, H. al., "Synthesis and Biological Evaluation of Novel Bisbenzimidazoles as Escherichia coli Topoisomerase IA Inhibitors and Potential Antibacterial Agents", J. Med. Chem. 2014, 57, 5238 - 5257; Chandrika, N. T. et al., "Synthesis and Investigation of Novel Benzimidazole Derivatives as Antifungal Agents", S. Bioorg. Med. Chem. 2016, 24, 3680 - 3686) in 75 ml of ethanol solution was added to 1.23 g (6.5 mmol) of Na2S2O5 in 3.0 ml of H2O. A white / grey precipitate formed.

[0155] The crude reduction mixture of diamine 10 was added to this suspension. The resulting orange - brown suspension was heated at 70 °C for 2 h. Complete conversion of the N - protected diamine 10 is shown by the DC control. Celite was added and the solvent was evaporated under vacuum. The solid residue was purified by sc - chromatography.

[0156] Ethyl acetate / MeOH gradient (100 / 0 → 80 / 20). 4.76 g of N - Boc - protected Hoechst 11 was isolated (yield 100%) R f : ca. 0.5 (ethyl acetate / MeOH / aqu. NH3: 70 / 28 / 2) HPLC: Luna 3μ, phenyl - hexyl, MeCN / H2O (98:2), R t 14.4 min C 33 H 39 N7O3 (581.7) MS - MALDI: 582.1 (M), 451.7 (M - BocHN - CH2)

[0157] 2'-(4-Ethoxyphenyl)-6-(4-aminoethyl-1-piperazinyl)-2,6'-bis-1H-benzimidazole (12)

[0158]

Chem.

[0159] N-Boc protected Hoechst amine 11 2.0 (3.44 mmol) was dissolved in 20 ml of EtOH and acidified with HCl / MeOH. After 24 hours at room temperature, the resulting precipitate was filtered and dried. 1.91 g of Hoechst amine 12·3 HCl was isolated (94%). Rf: approximately 0.5 (ethyl acetate / MeOH / aqu. NH3: 40 / 50 / 10) silica gel approximately 0.5 (CHCl3 / MeOH / aqu. NH3: 70 / 22 / 8) silica gel approximately 0.8 (CHCl3 / MeOH / aqu. NH3: 70 / 22 / 8) Al2O3 HPLC: Luna 3μ, phenyl-hexyl, MeCN / H2O (98:2), R t 11.8 minutes C 28 H 31 N7O (481.6) MS-MALDI: 482.7 (M+1), 451.5 (M-(N-CH2)) 11H-NMR (DMSO-d6): 1.37 (triplet, J = 7.0 Hz, 3H), 3.26 (multiplet, 4H), 3.38 (quartet, J = 7.0 Hz, 1H), 3.42 (quartet, J = 7.0 Hz, 1H), 3.48 (triplet, J = 7.0 Hz, 1H), 3.66 (doublet, J = 7.0 Hz, 1H), 3.90 (doublet, J = 7.0 Hz, 1H), 3.92 (multiplet, 1H), 4.17 (quartet, J = 7.0 Hz, 1H), 7.21 (doublet, J = 8.8 Hz, 2H), 7.22 (multiplet, 1H), 7.36 (doublet of doublet of doublet, J = 2.1, 5.8, 8.4 Hz, 1H), 7.72 (doublet, 3.5, 5.5 Hz, 1H), 7.97 (doublet, J = 8.5 Hz, 1H), 8.37 (multiplet, 1H), 8.38 (doublet, J = 8.7 Hz, 2H), 8.76 (singlet, 1H). 13 13C-NMR (DMSO-d6): 15.0, 33.9, 46.5, 51.3, 51.7, 53.3, 55.5, 64.2, 99.3, 99.5, 114.9, 115.8, 117.6, 124.1, 127.0, 130.4, 133.7, 138.2, 148.5, 149.0, 149.1, 153.2, 162.5.

[0160] (Example 7) Synthesis of 3-[4-(5-(4-methyl-1-piperazinyl)-(2,5'-bis-1H-benzimidazol-2-yl]-phenoxy-propylamine (L-Hoechst-C3)

[0161] [Chemical formula]

[0162] N-methoxy-N-methyl-3,4-diaminobenzamide (13)

[0163] [Chemical formula]

[0164] 2-(4-N-Boc-phenoxy-propylamine)-benzimidazole-5-carboxylic acid methoxy-methylamide (14)

[0165]

Chemical formula

[0166] 2.6 g (10.2 mmol) of Weinreb amide of 3,4-dinitrobenzoic acid was reduced under a hydrogen atmosphere to quantitatively obtain the corresponding N-methoxy-N-methyl-3,4-diaminobenzamide 13 (10.2 mmol). This reaction solution was used for the reaction with N-Boc-protected aldehyde 4 without any further purification.

[0167] A solution of 4.2 g (15 mmol) of N-Boc-protected aldehyde 4 in 100 ml of EtOH was added to a solution of 1.54 g (8.1 mmol) of Na2S2O5 in 3 ml of H2O. A yellow to gray precipitate was observed. The resulting suspension was further stirred at room temperature for 15 minutes. The filtered reaction solution of 10.2 mmol of N-methoxy-N-methyl-3,4-diaminobenzamide 13 in EtOH was added to this suspension, and the reaction mixture was heated at 65 °C for 30 minutes. Completion of the reaction was detected by TLC (ethyl acetate). The solvent was removed under vacuum, and the residue was purified by SC-chromatography with a CHCl3 / ethyl acetate gradient: 100 / 0 → 0 / 100. 4.0 g of Weinreb amide 14 was isolated (yield 86% over two reaction steps) HPLC: Luna 3μ, phenyl-hexyl, MeCN / H2O (98:2), R t 16.3 minutes C 24 H 30 N4O5 (454.2) MS-MALDI: 455.8 (M+1), 399 (M-56 (C4H8))

[0168] 2-(4-N-Boc-phenoxy-propylamine)-benzimidazole-5-carbaldehyde (15)

[0169]

Chem.

[0170] 1.3 g (2.9 mmol) of N-Boc wine levamide 14 was suspended in 24 ml of tetrahydrofuran and 8 ml of diethyl ether. This suspension was cooled to -80 °C, and 320 mg (9 mmol) of LiAlH4 was added. The resulting suspension was heated to -30 °C to -15 °C. The completion of the reaction was observed by TLC (ethyl acetate / hexane: 9 / 1). The reaction mixture was successively treated with ethyl acetate, MeOH, and saturated aqueous NH4Cl solution. The organic phase was separated, dried (Na2SO4), and the solvent was evaporated under vacuum. 1.05 g of N-Boc-aldehyde 15 was isolated by silica gel chromatography (yield 92%). This aldehyde 15 was used in the following oxidative cyclization without any further purification. HPLC: Luna 3μ, phenyl-hexyl, MeCN / H2O (98:2), R t 16.3 minutes C 22 H 25 N3O4 (395.5) MS-MALDI: 455.8 (M+1), 399 (M-56 (C4H8))

[0171] 2-(4-N-Boc-phenoxy-propylamine)-bis(benzimidazole) 6-(4-methylpiperazine) (16)

[0172]

Chem.

[0173] Piperazinylnitroaniline 3470 mg (2.0 mmol) was reduced under a hydrogen atmosphere to quantitatively obtain the corresponding piperazinyl diamine 5. This reaction solution was used in the reaction with N-Boc protected aldehyde 15 without any further purification.

[0174] 1.07 g (2.7 mmol) of N-Boc protected aldehyde 15 in 30 ml of EtOH was added to a solution of 385 mg (2.0 mmol) of Na2S2O5 in 2 ml of H2O. A gray precipitate formed. The suspension was stirred at room temperature for 30 minutes. The solution of piperazinyl diamine 5 was added to this suspension, and the reaction mixture was heated at 60 °C for 2 hours. Complete conversion of piperazionyl diamine 5 was shown by TLC control. Celite was added and the solvent was evaporated under vacuum. The solid residue was purified by silica gel chromatography. Gradient: CHCl3 / MeOH (100 / 0 → 95 / 5). HPLC: Luna 3μ, phenyl-hexyl, MeCN / H2O (98:2), R t 14.3 minutes C 33 H 39 N7O3 (581.3) MS-MALDI: 582.1 (M+1) 1 1H-NMR (DMSO-d6): 1.38 (s, 9H), 1.87 (septet, J = 6.6 Hz, 2H), 2.26 (s, 3H), 2.54 (m, 3H), 3.12 (tr, J = 6.6 Hz, 2H), 3.13 (d, J = 6.2 Hz, 2H), 3.31 (s, 3H), 4.07 (tr, J = 6.2 Hz, 2H), 6.91 (m, 2H), 7.11 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.1 Hz, 2H), 8.02 (d, J = 8.1 Hz, 2H), 8.13 (dd, J = 3.4, 8.3 Hz, 2H) 8.21 (m, ), 8.33 (m, 1H). 1313C-NMR (DMSO-d6): 28.2, 29.1, 36.9, 45.6, 49.9, 54.8, 65.5, 77.5, 108.8, 111.3, 114.8, 116.1, 118.6, 120.3, 121.0, 122.3, 124.3, 128.2, 135.3, 136.0, 144.2, 145.0, 147.6, 152.7, 155.6, 160.2.

[0175] 3-[4-(5-(4-Methyl-1-piperazinyl)-(2,5'-bis-1H-benzimidazol-2-yl]-phenoxy-propylamine (17) (Frau, S. et al., New J. Chem. 1995, 19, 873-6)

[0176]

Chemical Structure

[0177] N-Boc protected Hoechst 16 (813 mg, 1.4 mmol) was dissolved in 5 ml of MeOH. This solution was acidified with HCl / MeOH. After 24 hours at room temperature, complete deprotection was detected by TLC. The product crystallized in the reaction mixture. 400 mg was isolated by filtration (yield 60%). R f : about 0.5 CHCl3 / MeOH / H2O NH3 : 2 / 8 / 0.5 HPLC: Luna 3μ, phenyl-hexyl, MeCN / H2O (98:2), R t : 14.3 minutes C 28 H 31 N7O (481.6) MS-MALDI: 481.7 11H-NMR (DMSO-d6): 2.09 (quintet, J = 6.4 Hz, 2H), 2.83 (d, J = 3.6 Hz, 3H), 3.00 (sextet, J = 5.7 Hz, 2H), 3.22 (m, 4H), 3.53 (m, J = 8.4 Hz, 2H), 3.89 (m, J = 9.4 Hz, 2H), 4.23 (tr, J = 6.1 Hz, 2H), 7.21 (d, J = 2.0 Hz, 1H), 7.24 (d, J = 9.1 Hz, 2H), 7.35 (dd, J = 2.1, 9.0 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 8.38 (d, J = 8.6 Hz, 1H), 8.42 (d, J = 8.7 Hz, 2H), 8.77 (s, 1H), 13 13C-NMR (DMSO-d6): 26.9, 36.1, 41.9, 46.2, 52.0, 65.2, 98.9, 113.8, 114.2, 114.4, 115.2, 115.3, 117.2, 117.9, 118.7, 123.7, 126.2, 129.9, 133.2, 147.9, 148.7, 152.7, 161.7.

[0178] (Example 8) Binding of double-stranded DNA to agarose beads coupled with an affinity ligand YO-C3, TO-C3, TO-C6, MCMB, Mono-Hoechst-C3, L-Hoechst-C3 and R-Hoechst-C2 were synthesized as described above and coupled with agarose beads. Coupling of the functionalized agarose beads to the affinity ligand was achieved by the synthetic route shown in Table 3 (Table 3).

[0179] [Table 3]

[0180] Next, each of the coupled agarose-affinity ligands termed resins was tested for binding ds (double-stranded) DNA according to the following procedure. 100 μL of resin was placed into a mini spin column and equilibrated with a buffer (0.05 M TRIS, 0.5 M NaCl, pH 7.1). 200 μL of a ds DNA solution (Invitrogen, cat. No. 15634-017, LOT 1885913) was added at room temperature (RT) to create a suspension. The suspension was incubated at room temperature for 90 minutes and mixed with a vortexer every 30 minutes. After 90 minutes, the supernatant was removed by centrifugation, the liquid was collected, and measured at 260 nm using a UV light meter to detect the amount of DNA present in the liquid that did not bind to the resin. The resin was washed once with the buffer. The wash solution was also measured. In most cases, only a small amount of DNA was detected.

[0181] Next, if 100% of the DNA binds to the resin, the absorption at 260 nm becomes zero. Conversely, if the DNA does not bind to the resin at all, the absorption becomes 100%. Therefore, the amount of DNA bound to the resin can be measured based on the UV absorption of the liquid. The results are shown in Table 4 and Figure 6. Figure 6 is a bar graph showing the binding of ds DNA to the tested resin obtained by loading a solution having approximately 11 - 13 μg of ds DNA in the spin column onto 100 μl of the gel.

[0182]

Table 4

[0183] Table 5 presents more information regarding the compounds and sources of the products used in this experiment.

[0184]

Table 5

[0185] As shown in Figure 7, YO-C3, TO-C3, TO-C6, MCMB, Mono-Hoechst-C3, L-Hoechst-C3, and R-Hoechst-C3 successfully bound to ds DNA and were separated from the sample. PEI was included as a positive control. PEI is an imine polymer with positively charged amino groups and has a natural ionic interaction with DNA. Mimetic Yellow 2, Mimetic Orange 1, Mimetic Red 2, Mimetic Red 3, Mimetic Blue 1, Mimetic Blue 1M, Mimetic Blue SA HL, Mimetic Blue SA HL, Mimetic Blue 2 A6XL, and Mimetic Blue AP A6XL are dyes that are not intercalators for DNA. These were included as negative control dyes. Zetarose Mimetic Blue Dye 1FF, which has a higher affinity for DNA than other non-intercalating dyes simultaneously, was also included as a negative control. 6% of ds DNA bound to the unmodified agarose beads. This is the effect of non-specific binding. Therefore, this data shows that the intercalating small molecules bound to DNA successfully and selectively.

[0186] (Example 9) Effect on DNA binding of different dye ligand densities The pigment ligand density represents the number or amount per volume of the solid beads. The higher the density, the more DNA is predicted to be able to bind to the coupled surface - affinity ligand. This theory was tested by performing the procedure described in Example 8 using resins with different densities shown in Table 3 (Table 3). The results are shown in Table 6 (Table 6) - Table 10 (Table 10) and graphed in Figures 7 - 11. Figures 7, 8, 9, 11 show the binding of ds DNA to the tested resins using a loading solution with approximately 12 μg of ds DNA per 100 μl of gel in a spin column. Figure 10 shows the binding of ds DNA to agarose coupled with Mono - Hoechst - C3 resin using a loading solution with approximately 18 μg of ds DNA per 100 μl of gel in a spin column. In all of these figures, the amount of ds DNA binding to the resin is compared to the amount of binding to the control agarose beads.

[0187] [Table 6]

[0188] [Table 7]

[0189] [Table 8]

[0190] [Table 9]

[0191] [Table 10]

[0192] (Example 10) DNA Recovery Recovery of ds DNA from the prepared and coupled surface-affinity ligands shown in Table 3 and Table 4 was attempted using the following various different solvents: 1) 4 M NaCl, 2) 4 M NaCl and 95 °C, 3) pH 2 (0.1 M glycine, pH 2.0), and 4) pH 10 (0.2 M Na2CO3, pH 10).

[0193] Tables 11 to 14 show the amounts (%) of ds DNA isolated and recovered from each resin using different recovery methods and solvents.

[0194] [Table 11]

[0195] [Table 12]

[0196] [Table 13]

[0197] [Table 14]

[0198] (Example 11) Binding of double-stranded (ds) and single-stranded (ss) DNA Binding assays were performed using DNA (about 67 μg / mL for ds DNA, 0.2 mL; about 166 μg / mL for ss DNA, 0.2 mL) and 100 μL of resin. A number of non-intercalating dyes (Mimetic Yellow 2, Mimetic Orange 1, Mimetic Red 2, Mimetic Red 3, Mimetic Blue 1, Mimetic Blue 1M, Mimetic Blue SA HL and Mimetic Blue AP A6XL), unmodified agarose beads, and MCMB were tested for binding. The positive control was MCMB. For ds DNA, samples were loaded in a spin column using a buffer with 13.6 μg of ds DNA per 100 μL of gel. For ss DNA, samples were loaded in a spin column using a buffer with 22.1 μg of ss DNA per 100 μL of gel. Data are shown in Figures 12 and 13. As shown, none of the Astrea Biosciences Mimetic resins bound to ss DNA, and binding to ds DNA was negligible, while MCMB bound successfully to both ss DNA and ds DNA.

[0199] All of the non-MCMB resins had "negative" amounts of DNA binding and thus there may have been an error in the calibration. Negative results are reported as "zero" in this specification. Bound DNA was calculated by subtracting the eluted DNA from the measured mass of DNA in the loaded sample. This difference is the bound DNA. Measurements are all based on absorbance at 260 nm and calculation of the mass of DNA using Beer's law.

[0200] (Example 12) Selective non-binding of proteins To demonstrate the selective non-binding to the coupled agarose-affinity ligand, a DNA binding assay was performed using the model protein albumin. As positive controls, PEI and Astrea Biosciences' Mimetic resin were used since they are known to bind albumin. The experiment was conducted using 0.2 ml of 1 mg / ml albumin, 0.1 ml of resin, an incubation time of 90 minutes, room temperature, and 50 mM Tris / 0.5 M NaCl buffer.

[0201] Figure 14 is a graph showing the binding of albumin by the selected resins as a result of the experiment. Binding of albumin to the agarose-affinity ligand coupled to TO-C3, the agarose-affinity ligand coupled to TO-C6, and the agarose-affinity ligand coupled to MCMB was not shown. The minimum binding of 0.4 mg of albumin per 1 mL of resin was shown for YO-C3. Almost all of the albumin present bound to the positive control PEI (1.8 mg of albumin per 1 mL of resin). Why the Mimetic resin did not result in a binding value similar to that of PEI is unclear.

[0202] This experiment demonstrates that it is possible not to bind proteins using the affinity ligands of the present disclosure. However, appropriate selectivity depends on the proteins present in the sample and the use of the correct affinity ligand.

[0203] Although certain presently contemplated preferred embodiments of the invention are described, it will be understood by those skilled in the art that changes and modifications can be made to these without departing from the spirit of the invention. Also, all such changes and modifications are intended to fall within the true scope of the invention.

Claims

Claim 1 A method for separating a target polymer from a sample, comprising: a. selecting an affinity ligand that binds to the target polymer; b. binding the affinity ligand to a surface to create a coupled surface-affinity ligand; c. placing the coupled surface-affinity ligand in a container; d. introducing a sample containing the target polymer into the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time such that the target polymer binds to the affinity ligand; e. separating the coupled surface-affinity ligand to which the target polymer is bound from the sample from which the target polymer has been removed; wherein the target polymer is a nucleic acid or any fragment thereof, step a) comprises i. selecting an intercalator, minor groove binder, major groove binder, or any combination thereof that binds to the target polymer; ii. modifying the intercalator, minor groove binder, major groove binder, or combination thereof to contain a linker group to create an affinity ligand capable of binding to a surface, wherein the linker group is a reactive linker comprising a spacer ending in an epoxy group, carboxy group, halide group, or amino group; wherein the affinity ligand is a modified bisbenzimide, a modified cyanine of benzothiazole-quinoline, or a modified cyanine of benzoxazole-quinoline. Claim 2 The method of claim 1, wherein the target polymer is double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, single-stranded messenger RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), any fragment thereof, or any combination thereof. Claim 3 The method of claim 1, wherein the target polymer is DNA, RNA, any fragment thereof, or any combination thereof. Claim 4 The method of claim 1, wherein the target polymer is double-stranded DNA or double-stranded RNA. Claim 5 f. collecting an eluate that does not contain the target polymer, or f. eluting and recovering the target polymer from the coupled surface-affinity ligand; The method of claim 1, further comprising. Claim 6 The method of claim 1, wherein the spacer contains 1 to 30 atoms. Claim 7 The affinity ligand is as follows: 3-[4-(5-(4-methyl-1-piperazinyl)-(2,5'-bis-1H-benzimidazol-2-yl]-phenoxy-ethylamine (L-Hoechst-C2), 3-[4-(5-(4-methyl-1-piperazinyl)-(2,5'-bis-1H-benzimidazol-2-yl]-phenoxy-propylamine (L-Hoechst-C3), 3-[4-(6-(4-methyl-1-piperazinyl)-1H-benzimidazol-2-yl]-phenoxy-propylamine (Mono-Hoechst-C3), or 2'-(4-ethoxyphenyl)-6-(4-aminoethyl-1-piperazinyl)-2,6'-bis-1H-benzimidazole (R-Hoechst-C2) The method according to claim 1, which is a modified bisbenzimide selected from the above.

8. The affinity ligand is as follows: 1-(3-aminopropyl)-4-{[3-methyl-2,3-dihydro-1,3-benzoxazol-2-ylidene]methyl}quinolin-1-ium chloride (YO-C3), 1-(3-aminopropyl)-4-{[3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]methyl}quinolin-1-ium chloride (TO-C3), 【Chemical 1】 and TO-C6 [Chemical 2] The method according to claim 1, which is a modified cyanine of benzothiazole-quinoline or a modified cyanine of benzoxazole-quinoline selected from the above, and the spacer contains 1 to 30 atoms.

9. The method according to claim 1, wherein the surface is a solid surface, and the solid surface is beads, a membrane, particles, a mesh, a polymer, glass, metal, ceramic, silica, polysaccharide, or a monolith.

10. The method according to claim 9, wherein the solid surface contains a functionalized group, and the functionalized group contains an epoxy group, a carboxy group, an aldehyde group, or an amino group.

11. The method according to claim 9, wherein the solid surface is amino-agarose beads, or The method according to claim 9, wherein the solid surface is an aldehyde membrane.

12. The method according to any one of claims 1 to 11, which is used in chromatography.

13. The method according to claim 1, for isolating and extracting DNA from a sample containing DNA and protein, comprising: selecting an affinity ligand that binds to f.DNA; g. A step of binding an affinity ligand to a surface to create a coupled surface-affinity ligand; h. A step of placing the coupled surface-affinity ligand in a container; i. A step of introducing a sample to the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, wherein DNA binds to the affinity ligand; j. A step of separating the coupled surface-affinity ligand bound to DNA from the sample from which the DNA has been removed comprising, a method, wherein the target macromolecule is a nucleic acid and the target macromolecule is DNA.

14. The method according to claim 13, wherein the DNA is double-stranded.

15. The method according to claim 1, for isolating and extracting RNA from a sample containing RNA and protein, k. A step of selecting an affinity ligand that binds to RNA; l. A step of binding the affinity ligand to a surface to create a coupled surface-affinity ligand; m. A step of placing the coupled surface-affinity ligand in a container; n. A step of introducing a sample to the coupled surface-affinity ligand and incubating the coupled surface-affinity ligand with the sample for a residence time, wherein RNA binds to the affinity ligand; o. A step of separating the coupled surface-affinity ligand bound to RNA from the sample from which the RNA has been removed comprising, a method, wherein the target macromolecule is a nucleic acid and the target macromolecule is RNA.

16. The method according to claim 15, wherein the RNA is double-stranded.

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