Nucleic acid end detection method

Abstract

The present invention relates to a method for detecting nucleic acid ends within a nucleic acid-containing biological material. The present invention also relates to a kit for detecting nucleic acid ends within a biological material, and related uses of the kit. In addition, the present invention relates to a method for evaluating the efficacy of a therapeutic agent.

Description

Technical Field

[0001] The present invention relates to a method for detecting nucleic acid termini in biological materials containing nucleic acids. The present invention also relates to a kit for detecting nucleic acid termini in biological materials, and related uses of the kit. In addition, the present invention relates to a method for evaluating the efficacy of therapeutic agents.

Background Art

[0002] Since the concept of synthetic lethality was proposed in the context of the DNA damage response, the homologous recombination (HR) status of cancer cells has been a focus of attention for researchers and clinicians. Functional homologous recombination repair deficiency (HRD) can be defined by a low score of RAD51. For example, a low score of RAD51 has been revealed as an indicator of patient prognosis and response to therapeutic agents used to treat triple-negative breast cancer. A low score of RAD51 is also used to evaluate the therapeutic efficiency of PARP inhibitors. Although different solutions for detecting RAD51 (e.g., RAD51 immunofluorescence (IF)) exist, no functional biomarker that can evaluate the proficiency of RAD51-driven HR has been found.

[0003] Mismatch repair (MMR) is considered one of the fundamental pathways for monitoring genomic stability, and it has been revealed that mutations in genes encoding important MMR proteins (e.g., PMS1, PMS2, or MLH1) promote the development of cancer. On the other hand, inactivation of MMR components in cancer cells leads to an increase in neoantigen formation, and as a result, exposes tumors to the immune system. Therefore, therapeutic strategies targeting the inhibition of MMR are under development, and methods for reporting the status of MMR are still lacking.

[0004] WO2019 / 035727A1 describes a method for detecting DNA ends. This method relies on a molecule, such as BrdU, directly binding to a DNA end, followed by a binding molecule (e.g., an antibody) binding to the molecule bound to the DNA end. In this method, the binding molecule conjugates with an oligonucleotide, which can hybridize with further oligonucleotides to form a circular structure. This circular structure provides a template for rolling circle amplification. Once amplified, the amplification product is detectable by detection methods known in the art, such as fluorescence microscopy. However, this method cannot directly detect nucleic acid-binding proteins involved in nucleic acid (e.g., DNA) damage response, mismatch repair, and homologous recombination.

[0005] Thus, there is a need for more precise solutions for detecting proteins involved in nucleic acid (e.g., DNA) damage response and repair, mismatch repair, and homologous recombination. [Overview of the project]

[0006] The inventors have modified the method of WO2019 / 035727A1 (referred to as "STRIDE") to enable the detection of nucleic acid-binding proteins (such as PMS1, PMS2, MLH1, RAD51, or RPA70) bound to nucleic acids at or near the nucleic acid ends (e.g., DNA ends) in biological materials. The inventors have found a method for accurately detecting and quantifying proteins involved in nucleic acid (e.g., DNA) damage response and repair, mismatch repair, and homologous recombination. Accordingly, the solution provides a method for simultaneously detecting nucleic acid ends and nucleic acid-binding proteins bound to nucleic acids at those ends. In particular, the method of the present invention includes detecting cytosolic nucleic acids (especially DNA) that can be bound, for example, by cyclic GMP-AMP synthase (cGAS).

[0007] In one embodiment of the present invention, the present invention relates to a method for detecting nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating biological material with proteinase; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid end, and the other binding molecule binds to a nucleic acid binding protein at or near the nucleic acid end; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. This provides a method that includes [something].

[0008] Incubating biological materials with proteinase improves the detection of nucleic acid-binding proteins within the biological materials (compared to situations where proteinase is not used). Specifically, incubating biological materials with proteinase increases accessibility to nucleic acid ends without significantly affecting the levels of nucleic acid-binding proteins within the biological materials.

[0009] While not bound by theory, increased accessibility to nucleic acid ends can minimize steric hindrance in the localization of detectable DNA ends.

[0010] Embodiments and aspects referring to methods for detecting nucleic acid ends in biological materials should be understood to also refer to methods for detecting nucleic acid-binding proteins that bind to nucleic acids at or near the nucleic acid ends. Thus, embodiments and aspects described herein can be understood to refer to methods for detecting nucleic acid-binding proteins in biological materials containing nucleic acid-binding proteins.

[0011] The step of incubating the biological material with proteinase may be carried out under conditions such as using proteinase K at a concentration of about 7 μg / mL (e.g., 7.1 μg / mL) to increase accessibility to the nucleic acid ends to a level achievable at a temperature of about 22°C (i.e., room temperature) in less than 2 minutes. Incubation with proteinase may be carried out by referring to any one of Examples 1-3.

[0012] The present invention is a method for detecting nucleic acid ends in a biological material containing nucleic acids, wherein a) A process of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at approximately 22°C (i.e., room temperature) in less than 2 minutes using proteinase K at a concentration of 6 μg / mL to 8 μg / mL; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. This provides a method that includes [something].

[0013] The present invention is a method for detecting nucleic acid ends in a biological material containing nucleic acids, wherein a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 2 minutes using proteinase K at a concentration of approximately 7 μg / mL (e.g., 7.1 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. This provides a method that includes [something].

[0014] The present invention relates to a method for detecting single-stranded nucleic acid ends in nucleic acid-containing biological materials, a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 60 seconds using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It includes, where the nucleic acid-binding protein binds to the end of a single-stranded nucleic acid. Provide a method.

[0015] The present invention relates to a method for detecting double-stranded nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 2 minutes using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., approximately 7 μg / mL); b) adding a nucleic acid-binding molecule to the biological material under conditions such that the nucleic acid-binding molecule binds to the nucleic acid terminus within the biological material; c) adding two different binding molecules to the biological material under conditions such that one binding molecule binds to the nucleic acid-binding molecule bound to the nucleic acid terminus and the other binding molecule binds to the nucleic acid-binding protein that binds to the nucleic acid at the nucleic acid terminus; and d) detecting the nucleic acid terminus within the biological material by detecting the co-localization of the nucleic acid-binding protein and the nucleic acid terminus through the binding of the two different binding molecules comprising, wherein the nucleic acid-binding protein binds to the double-stranded nucleic acid terminus, to provide a method.

[0016] The present invention is a method for detecting a nucleic acid terminus within a biological material containing a nucleic acid, wherein a) incubating the biological material with a protease for less than 2 minutes; b) adding a nucleic acid-binding molecule to the biological material under conditions such that the nucleic acid-binding molecule binds to the nucleic acid terminus within the biological material; c) adding two different binding molecules to the biological material under conditions such that one binding molecule binds to the nucleic acid-binding molecule bound to the nucleic acid terminus and the other binding molecule binds to the nucleic acid-binding protein that binds to the nucleic acid at the nucleic acid terminus; and d) detecting the nucleic acid terminus within the biological material by detecting the co-localization of the nucleic acid-binding protein and the nucleic acid terminus through the binding of the two different binding molecules to provide a method.

[0017] The present invention is a method for detecting a single-stranded nucleic acid terminus within a nucleic acid-containing biological material, a) incubating the biological material with a protease for less than 60 seconds; b) adding a nucleic acid-binding molecule to the biological material under conditions such that the nucleic acid-binding molecule binds to the nucleic acid terminus within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It includes, where the nucleic acid-binding protein binds to the end of a single-stranded nucleic acid. Provide a method.

[0018] The present invention relates to a method for detecting double-stranded nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating the biological material with proteinase for less than 2 minutes; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It includes, where the nucleic acid-binding protein binds to the ends of the double-stranded nucleic acid. Provide a method.

[0019] In all aspects and embodiments described herein, preferably, the nucleic acid comprises or consists of DNA. In all aspects and embodiments described herein, preferably, the nucleic acid ends are DNA ends. In all aspects and embodiments described herein, the proteinase is preferably proteinase K. In all aspects and embodiments described herein, preferably, the nucleic acid-binding protein and the nucleic acid-binding molecule are not the same molecule. Thus, it is preferable that the nucleic acid-binding protein and the nucleic acid-binding molecule are different molecules.

[0020] In one embodiment of the present invention, the present invention is a kit for detecting nucleic acid ends in biological materials, a) Nucleic acid binding molecules; b) A binding molecule that binds to the nucleic acid binding molecule; and c) Binding molecules that bind to nucleic acid-binding proteins We provide a kit that includes this.

[0021] In one embodiment of the present invention, the present invention is a kit for detecting nucleic acid ends in biological materials, a) Nucleic acid binding molecules; b) A monoclonal antibody that binds to the nucleic acid-binding molecule; and c) Monoclonal antibodies that bind to nucleic acid-binding proteins We provide a kit that includes this. Preferably, the kit further comprises a proteinase (e.g., proteinase K).

[0022] In one embodiment, the present invention relates to a method for evaluating the effectiveness of a therapeutic agent, a) A step of performing the detection method described herein on a sample obtained from a subject before administering the therapeutic agent; b) A step of performing the detection method described herein on a sample obtained from a subject after administering a therapeutic agent; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where, if the amount of nucleic acid ends detected in step a) is greater than that detected in step b), the therapeutic agent is effective). This includes, where steps a) and b) can be carried out in any order. Provide a method. Preferably, the therapeutic agent targets PMS1, PMS2, or MLH1.

[0023] In one embodiment, the present invention relates to a method for evaluating the effectiveness of a therapeutic agent, a) A step of performing the detection method described herein on a sample obtained from a subject before administering the therapeutic agent; b) A step of performing the detection method described herein on a sample obtained from a subject after administering a therapeutic agent; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where, if the amount of nucleic acid ends is greater in step b) than in step a), the therapeutic agent is effective). This includes, where steps a) and b) can be carried out in any order. Provide a method. Preferably, the therapeutic agent targets RPA (e.g., RPA70) or RAD51.

[0024] In one embodiment, the present invention relates to a method for evaluating nucleic acid damage induced by a drug, a) The step of performing the detection method described herein on a sample obtained from a subject before administering or being exposed to the drug; b) The step of performing the detection method described herein on a sample obtained from a subject that has been administered a drug or has been exposed to a drug; c) A step of comparing the amounts of nucleic acid ends detected in steps a) and b) (where, if the amount of nucleic acid ends is greater in step b) than in step a), the drug induces nucleic acid damage). This includes, where steps a) and b) can be carried out in any order. Provide a method.

[0025] In one embodiment, the present invention provides a method for predicting the response of a subject diagnosed with cancer to anti-cancer therapy, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by performing the detection method described herein; and (ii) predicting the response of the subject to anti-cancer therapy based on the level of nucleic acid-binding proteins measured in the sample.

[0026] In one embodiment, the present invention provides a method for selecting a customized therapy for a subject diagnosed with cancer, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by performing the detection method described herein; and (ii) selecting a customized therapy for the subject based on the level of nucleic acid-binding proteins measured in the sample.

[0027] In one embodiment, the present invention provides a method for classifying subjects diagnosed with cancer into patient cohorts, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subjects by performing the detection method described herein; and (ii) classifying the subjects diagnosed with cancer into patient cohorts based on the level of nucleic acid-binding proteins measured in the sample.

[0028] In one embodiment, the present invention provides a method for predicting whether a tumor derived from a subject diagnosed with cancer is capable of DNA repair by homologous recombination, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by carrying out the detection method described herein; and (ii) predicting, based on the level of nucleic acid-binding proteins measured in the sample, whether the tumor derived from the subject diagnosed with cancer can be repaired by homologous recombination.

[0029] In one embodiment, the present invention provides a method for evaluating the state of mismatch repair pathways in a sample containing tumor cells obtained from a subject, comprising: (i) measuring the level of nucleic acid-binding proteins in tumor cells by carrying out the detection method described herein; and (ii) evaluating the state of mismatch repair pathways based on the level of nucleic acid-binding proteins measured in tumor cells.

[0030] In one embodiment, the present invention provides a pharmaceutical for use in the treatment of cancer in a subject, wherein the subject is identified as a responder to the pharmaceutical by the method described herein.

[0031] Each aspect or embodiment described herein can be combined with any other aspect or embodiment unless expressly indicated otherwise. In particular, any feature indicated as preferred or advantageous can be combined with any other feature indicated as preferred or advantageous.

[0032] The inventors have unexpectedly discovered that biological materials containing nucleic acids and nucleic acid-binding proteins (e.g., cells) can be subjected to proteinase treatment to increase accessibility to nucleic acid ends without significantly affecting the levels of nucleic acid-binding proteins within the biological material. Increasing accessibility to nucleic acid ends facilitates the binding of nucleic acid-binding molecules to the nucleic acid ends, thereby improving the detection level of nucleic acid ends and nucleic acid-binding proteins within the biological material. Thus, the inventors have found a more accurate and sensitive method for detecting nucleic acid-binding proteins (and co-localized nucleic acid ends) within biological materials. [Modes for carrying out the invention]

[0033] Unless otherwise specified herein, scientific and technical terms used in connection with the present invention shall have meanings that are commonly understood by those skilled in the art. However, the meaning and scope of such terms should be clear, and in any event of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions.

[0034] While singular prepositions such as "a," "an," and "the" are often used for convenience, it should be understood that all singular forms include the plural unless explicitly stated or otherwise indicated by the context. The words "include," "contain," and "have" are comprehensive and mean that there may be additional components beyond those listed. Furthermore, it should be understood that all documents presented in this disclosure, including academic papers, books, patents, and technical documents, are incorporated herein in their entirety by attribution for all purposes.

[0035] In numerical data as used herein, the term "approximately" refers to a value within 10% (i.e., ±10%) of the underlying parameter, and the use of the term "approximately" at the beginning of a series of numbers modifies each of the numbers (i.e., "approximately 1, 2, and 3" means approximately 1, approximately 2, and approximately 3). For example, a temperature of "approximately 25°C" may encompass temperatures from 22.5°C to 27.5°C (including both ends).

[0036] The present invention provides a method for detecting nucleic acid ends (e.g., DNA ends) in a biological material containing nucleic acids (e.g., DNA). The present invention also provides a method for detecting nucleic acid-binding proteins in a biological material containing nucleic acid-binding proteins. The method depends on the presence of the nucleic acid-binding protein either i) near the nucleic acid ends or ii) bound to the nucleic acid ends. The method depends on detecting the event of colocalization between the nucleic acid-binding protein and the nucleic acid ends within the biological material. It is preferable that the nucleic acid-binding protein is found within the biological material. That is, the method depends on the presence of endogenous nucleic acid-binding proteins in the biological material.

[0037] The requirement that "the nucleic acid-binding protein binds to the nucleic acid at its nucleic acid end" means that the nucleic acid-binding protein binds directly or indirectly to the nucleic acid end, in close proximity to it, so that colocalization between the nucleic acid-binding protein and the nucleic acid end can be detected by the method described herein. Sufficient proximity typically means that the nucleic acid end is within 100 nm, preferably within 50 nm, and most preferably within 40 nm. The nucleic acid-binding protein may bind indirectly to the nucleic acid at its nucleic acid end, for example, as part of a (multiprotein) complex that provides sufficient proximity to the nucleic acid end to enable detection. The nucleic acid-binding protein may also bind to a cytoplasmic nucleic acid fragment, for example, in the case of cGAS. As will be readily apparent to those skilled in the art, the term "at the nucleic acid end" as used herein includes nucleic acid residues at the ends of a nucleic acid chain. This includes gaps within the nucleic acid chain. Preferably, the nucleic acid end is the result of nucleic acid damage, homologous recombination, or mismatch repair.

[0038] In the context of nucleic acid-binding proteins, the term "endogenous" encompasses nucleic acid-binding proteins found within biological materials. Thus, these proteins are not added to the biological material as part of the method or kit described herein. Endogenous nucleic acid-binding proteins are naturally found (i.e., naturally present) within biological materials. Thus, biological materials (e.g., cells) may contain nucleic acids and nucleic acid-binding proteins prior to the proteinase treatment step. Endogenous nucleic acid-binding proteins are not added to the biological material by the user of the method or kit.

[0039] As used herein (in the context of co-localization between nucleic acid ends and nucleic acid-binding proteins), detection based on "co-localization" means that the presence of a nucleic acid end and a nucleic acid-binding protein is detected based on their proximity to each other. This typically involves a single detection (e.g., fluorescence) signal that occurs only when the nucleic acid end and the nucleic acid-binding protein are in close proximity to each other, i.e., when the nucleic acid-binding protein is bound to the nucleic acid at its nucleic acid end. In the absence of this close proximity, no signal is generated. The nucleic acid-binding protein detected according to the present invention may be such a protein as one that is involved in the detection or repair of nucleic acid damage (i.e., damage resulting at a detectable nucleic acid end, including cytoplasmic chromatin fragments).

[0040] Nucleic acid-binding proteins may bind to nucleic acids at their nucleic acid terminals either directly (i.e., by direct association with the nucleic acid) or indirectly (i.e., by forming a complex molecule with at least one other molecule (e.g., a protein) that directly binds to the nucleic acid and thus acts as an adapter molecule for the indirect binding of the nucleic acid-binding protein).

[0041] By screening a series of experimental parameters, the inventors found optimal conditions for detecting the co-localization of nucleic acid-binding proteins with nucleic acid ends (co-localization where nucleic acid-binding proteins bind directly or indirectly, as defined herein). Specifically, the inventors found that proteinase treatment (before the detection step) increases accessibility to nucleic acid ends in the biological material without significantly affecting the level of the nucleic acid-binding protein to be detected. Preferably, the proteinase is serine proteinase (particularly proteinase K). The inventors found that treating with proteinase at a concentration of 6 μg / mL to 8 μg / mL (e.g., about 7 μg / mL) for less than 2 minutes (preferably 90 seconds or less) at a temperature of about 22°C (i.e., room temperature) achieves the optimal combination for improving accessibility to nucleic acid ends without significantly affecting the level of the nucleic acid-binding protein to be detected. Those skilled in the art will understand that these parameters can be varied, for example, by lowering the proteinase concentration and extending the processing time, or by increasing the proteinase concentration and shortening the processing time. When detecting single-stranded nucleic acid ends in biological material, a shorter proteinase treatment is preferable compared to detecting double-stranded nucleic acid ends. The methods described herein may include a step of incubating the biological material with proteinase to raise accessibility to the nucleic acid ends to a level that can be achieved by using proteinase (e.g., proteinase K) at a concentration of 6 μg / mL to 8 μg / mL (e.g., about 7 μg / mL) for less than 2 minutes (preferably 90 seconds or less) (without significantly affecting the level of nucleic acid-binding protein to be detected). If the nucleic acid ends are single-stranded, the method described herein may include the step of incubating the biological material with a proteinase and raising the accessibility to the nucleic acid ends to a level that can be achieved by using a proteinase (e.g., proteinase K) at a concentration of 6 μg / mL to 8 μg / mL (e.g., about 7 μg / mL) for less than 90 seconds (preferably about 30 seconds) (without significantly affecting the level of nucleic acid-binding protein to be detected).Preferably, the method is carried out under conditions such that the level of the nucleic acid-binding protein to be detected does not decrease significantly.

[0042] In this specification, as used in the context of nucleic acid-binding protein levels, the expressions “without significant effect,” “not significantly reduced,” or “without significantly reducing” mean that after treatment with a proteinase, the detectable level of nucleic acid-binding protein remains substantially the same as before treatment. For example, the detectable level of nucleic acid-binding protein after treatment may be 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, or at least 50% of the level before treatment. Preferably, the level of nucleic acid-binding protein remains detectable, and detection is better than without proteinase treatment because the background protein is reduced.

[0043] The biological material may contain nucleic acids (e.g., DNA). The biological material may also contain nucleic acid-binding proteins (e.g., DNA-binding proteins).

[0044] The present invention is a method for detecting nucleic acid ends in a biological material containing nucleic acids, wherein a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein bound to the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. This provides a method that includes [something].

[0045] The present invention also provides a method for detecting nucleic acid-binding proteins in biological materials containing nucleic acid-binding proteins, a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein bound to the nucleic acid terminal; and d) A process for detecting nucleic acid-binding proteins in biological materials by detecting the co-localization of nucleic acid-binding proteins and nucleic acids through the binding of two different binding molecules. This provides a method that includes [something].

[0046] As discussed herein, nucleic acid-binding proteins can bind to nucleic acids either directly or indirectly.

[0047] Thus, a method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that is directly bound to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may include.

[0048] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein bound to the nucleic acid terminal via an adapter molecule (e.g., an adapter protein); and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may include.

[0049] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating a biological material with a proteinase to increase accessibility to the nucleic acid terminus to a level achievable by using a proteinase (e.g., proteinase K) for less than two minutes; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may include.

[0050] The present invention relates to a method for detecting single-stranded nucleic acid ends in nucleic acid-containing biological materials, a) A step of incubating the biological material with proteinase for less than 60 seconds; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It includes, where the nucleic acid-binding protein binds to the end of a single-stranded nucleic acid. Provide a method.

[0051] The present invention relates to a method for detecting double-stranded nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating the biological material with proteinase for less than 2 minutes; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It includes, where the nucleic acid-binding protein binds to the ends of the double-stranded nucleic acid. Provide a method.

[0052] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating a biological material with a proteinase to increase the accessibility to the nucleic acid ends to a level achievable by using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., about 7 μg / mL) for less than 2 minutes at a temperature of about 22°C (i.e., room temperature); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may include.

[0053] The present invention relates to a method for detecting single-stranded nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 60 seconds using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may also include, where the nucleic acid-binding protein binds to the end of a single-stranded nucleic acid. Provide a method.

[0054] The present invention relates to a method for detecting double-stranded nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 2 minutes using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may also include, where the nucleic acid-binding protein binds to the ends of the double-stranded nucleic acid. Provide a method.

[0055] Incubation of biological materials with proteinase may be carried out for less than 100 seconds, less than 90 seconds, less than 80 seconds, less than 70 seconds, or less than 60 seconds, preferably 90 seconds or less.

[0056] Thus, a method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating a biological material with proteinase to increase accessibility to the nucleic acid ends to a level achievable by using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., about 7 μg / mL) for 90 seconds or less at a temperature of about 22°C (i.e., room temperature); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may include.

[0057] Incubation of biological materials with proteinase can be carried out for 1 to less than 2 minutes, 15 to 90 seconds, 20 to 80 seconds, 25 to 70 seconds, or 30 to 60 seconds, with incubation of biological materials with proteinase being preferably carried out for 30 to 60 seconds.

[0058] The methods, kits, and uses described herein can focus on the detection of nucleic acid-binding proteins that specifically bind to single-strand nucleic acid ends (i.e., single-strand nucleic acid cleavage sites, e.g., those induced by DNA damage). When detecting nucleic acid-binding proteins that bind to single-strand nucleic acid cleavage sites (e.g., PMS2), the incubation may be carried out over a period of 1 to less than 90 seconds, 5 to 75 seconds, 10 to 60 seconds, or 15 to 45 seconds. Incubation is preferably carried out over a period of approximately 30 seconds.

[0059] The methods, kits, and uses described herein can focus on the detection of nucleic acid-binding proteins that specifically bind to the ends of double-stranded nucleic acids (i.e., double-stranded nucleic acid cleavage sites, e.g., those induced by DNA damage). When detecting nucleic acid-binding proteins that bind to double-stranded nucleic acid cleavage sites (e.g., RAD51 or RPA), the incubation may be carried out over a period of 1 second to less than 2 minutes, 10 seconds to 90 seconds, 20 seconds to 80 seconds, or 30 seconds to 70 seconds. Incubation is preferably carried out over a period of approximately 60 seconds.

[0060] For nucleic acid-binding proteins that specifically bind to single-strand nucleic acid ends (e.g., PMS1, PMS2, or MLH1), it is preferable to shorten the proteinase incubation time compared to double-strand nucleic acid ends, considering the smaller amount of these proteins present at the nucleic acid end site (e.g., nucleic acid damage). The proteinase incubation time is broadly related to the characteristics of the specific nucleic acid-binding protein; for example, in the case of double-strand breaks, multiple molecules of RPA and RAD51 are recruited to the nucleic acid end. This number is smaller when the PMS2 protein is recruited to the single-strand break site.

[0061] The incubation of the biological material with proteinase is carried out for at least 1 second, at least 5 seconds, preferably at least 10 seconds, and more preferably at least 15 seconds.

[0062] Thus, a method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 1 second to 2 minutes using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may include.

[0063] Thus, a method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating the biological material with proteinase to increase the accessibility to the nucleic acid ends to a level achievable by using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., about 7 μg / mL) for 15 to 90 seconds at a temperature of about 22°C (i.e., room temperature); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. It may include.

[0064] The incubation of the biological material with proteinase may be carried out for about 30 seconds or about 60 seconds. For the detection of single-stranded nucleic acid ends, the incubation step of the biological material with proteinase is preferably 90 seconds or less, 60 seconds or less, and more preferably about 30 seconds. For the detection of double-stranded nucleic acid ends, the incubation step of the biological material with proteinase is preferably less than 2 minutes, 90 seconds or less, and more preferably about 60 seconds.

[0065] The incubation of biological materials with proteinase may be carried out at temperatures of 10°C to 55°C, 15°C to 55°C, 20°C to 55°C, 10°C to 35°C, or 15°C to 30°C, preferably 20°C to 25°C. More preferably, the incubation of biological materials with proteinase (e.g., proteinase K) is carried out at a temperature of about 22°C (i.e., room temperature).

[0066] The proteinase may be in solution form. Preferably, the concentration of the proteinase is 0.1 μg / mL to 100 μg / mL, 1 μg / mL to 20 μg / mL, 3 μg / mL to 15 μg / mL, 5 μg / mL to 10 μg / mL, or 6 μg / mL to 8 μg / mL, preferably 6 μg / mL to 8 μg / mL. Preferably, the concentration is less than 10 μg / mL. The proteinase may be at a concentration of about 7 μg / mL. The solution may contain a buffer such as PBS. The solution may contain SDS (e.g., 0.2 to 1% SDS) and / or urea (e.g., 1 to 4 M urea). The solution may have a pH of 4.0 to 12.5, preferably pH 7.0 to 8.0. For example, if the solution contains PBS, the pH is about 7.4.

[0067] The inventors have found that incubating biological materials with proteinase K (i.e., at a concentration of 6-8 μg / mL for less than 2 minutes), as described herein, achieves more accurate detection of nucleic acid ends compared to using proteinase K at a concentration of 10 μg / mL for 2.5 minutes.

[0068] The proteinase may also be a broad-spectrum proteinase. The term "broad-spectrum proteinase" encompasses proteinases that have the ability to digest a wide range of native proteins. A particularly preferred example of a broad-spectrum proteinase is proteinase K. The primary cleavage site of proteinase K is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids, where the alpha-amino group is blocked. Any other proteinase having the same or similar mechanism of action is considered a broad-spectrum proteinase within the scope of the present invention.

[0069] The proteinase may be aspartate protease, glutamate protease, metalloprotease, cysteine ​​protease, serine protease, or threonine protease. Preferably, the protease is a serine protease such as proteinase K. In all aspects and embodiments described herein, the proteinase is preferably proteinase K.

[0070] The incubation of biological material with proteinase can be stopped by adding a proteinase inhibitor. Any suitable proteinase inhibitor may be used. For example, the proteinase inhibitor may be phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), or carbiochem. The proteinase inhibitor may be incubated with the proteinase (in contact with the biological material) for at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, or at least 5 minutes. For example, 1 mM PMSF may be used for 5 minutes to stop the action of proteinase. After incubation, the proteinase inhibitor can be removed by a washing step (e.g., using a buffer such as PBS). The incubation of biological material with proteinase can be stopped by at least one rinsing step.

[0071] The present invention is a method for detecting nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating the biological material with proteinase for less than 2 minutes; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. This provides a method that includes [something].

[0072] The biological material may be an animal, plant, protozoan, bacterium, or virus, or derived therefrom. The biological material may be a cell or tissue, or a fragment thereof. The biological material may include living cells or fixed cells. The method may include a fixation step before the step of incubating the biological material with a proteinase. The fixation step may include incubating the biological material with ethanol (e.g., 70% (v / v) ethanol). The fixation step may be carried out over a period of 1 to 24 hours, 2 to 12 hours, or 3 to 6 hours. Preferably, the fixation step is carried out over a period of at least 2 hours. The fixation step may be carried out over a period of at least 8 hours. The fixation step may be carried out at a temperature of -80°C to 30°C or -40°C to 25°C, preferably about -20°C. The method may further include a step of attaching the biological material to a solid support before the step of incubating the biological material with a proteinase. The step of attaching the biological material to a solid support may be carried out before or after the fixation step (if any). Preferably, the solid support is a hydrophilic solid support (i.e., a positively charged solid support and / or a negatively charged solid support). A hydrophilic solid support means a solid support comprising at least one hydrophilic outer surface. Thus, the hydrophilic surface is presented to the biological material, thereby resulting in the adhesion of individual cells to the solid support (via the hydrophilic surface). The hydrophilic solid support may be a slide or a coverslip. The hydrophilic properties of the solid support may be obtained by coating the solid support or from the composition of the solid support itself. A coating may provide a positively or negatively charged surface, but a positively charged surface is preferred. For example, the solid support may be a TOMO® slide or a poly-L-lysine coated slide, both of which provide a positively charged surface on the surface to which cells bind. In some embodiments, the solid support may be a silicate-uncoated glass slide. The biological material (e.g., cells) may be in a suspension state.

[0073] The process of attaching biological materials (e.g., cells) may be carried out at a temperature of 10°C to 40°C or 15°C to 37°C. For example, the process of attaching biological materials (e.g., cells) may be carried out at a temperature of 20°C to 27°C or 34°C to 40°C. The process of attaching biological materials (e.g., cells) may be carried out at a temperature of approximately 25°C or approximately 37°C. The process of attaching biological materials (e.g., cells) may be carried out over a period of 30 minutes to 4 hours, or 1 hour to 3 hours. Preferably, the process of attaching biological materials (e.g., cells) is carried out over a period of approximately 2 hours. The process of attaching biological materials (e.g., cells) may be carried out over a period of at least 30 minutes, at least 1 hour, at least 90 minutes, at least 2 hours, at least 2.5 hours, or at least 3 hours. The process of attaching biological materials (e.g., cells) may be carried out in a humidified chamber and / or a CO2 incubator. The process of attaching biological materials (e.g., cells) may be carried out under conditions such that the cells do not dry out during the attachment process. That is, the process of attaching biological materials (e.g., cells) ensures that the cells or components of the biological material remain substantially covered in liquid throughout the process.

[0074] In all embodiments described herein, the nucleic acid may be DNA and / or RNA, or may contain both, and preferably the nucleic acid is DNA. The nucleic acid end may be a DNA end, an RNA end, or a hybrid DNA / RNA end, and preferably the nucleic acid end is a DNA end.

[0075] The methods described herein have sufficient sensitivity and specificity to detect single-stranded and double-stranded nucleic acid ends. Thus, nucleic acid ends may be single-stranded ends and / or double-stranded ends. Nucleic acid ends may be single-stranded nucleic acid cleavage and / or double-stranded nucleic acid cleavage. Nucleic acid ends may be single-stranded gaps or single-stranded nicks. Nucleic acid ends may be double-stranded blunt-end cleavage or double-stranded 3'-overhang end cleavage.

[0076] The high sensitivity and / or specificity of this method makes it possible to detect single-stranded nucleic acid ends within cells. Thus, the nucleic acid ends may be single-stranded nucleic acid ends. The methods described herein depend on the addition of a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material.

[0077] The nucleic acid binding molecule used in the methods and kits described herein may be any molecule having the ability to bind to the ends of nucleic acid molecules. The nucleic acid binding molecule used in the methods and kits described herein may also be bound by binding molecules (e.g., antibodies or fragments thereof) that may (or may) attach to oligonucleotide molecules. Preferably, a first portion of the nucleic acid binding molecule or a first site on the nucleic acid binding molecule interacts with and / or binds to the nucleic acid ends, and a second portion of the nucleic acid binding molecule or a second site on it interacts with and / or binds to one of the binding molecules. (i) Halidelated nucleotides or nucleoside molecules such as BrdU, IdU, and CldU; (ii) DNA precursor analogs such as EdU (5-ethynyl-2'-deoxyuridine), F-ara-EdU, and 5-ethynyl-2'-deoxycytidine; (iii) Biotinylated nucleotide molecules; (iv) ADP-ribose molecule; (v) Protein molecule; (vi) A nucleotide or nucleoside molecule labeled with a label (wherein optionally, the label is selected from the group consisting of a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, or a biotin molecule). A selection may be made from the group consisting of the following:

[0078] Preferably, the nucleic acid binding molecule is a halogenated nucleotide or nucleoside molecule, a DNA precursor analog, and / or a biotinylated nucleotide molecule. Preferably, the nucleic acid binding molecule is not the same as the nucleic acid binding protein. The nucleic acid binding molecules may be of the same type, or may be of at least two, at least three, at least four, or at least five different types. For example, the nuclear nucleic acid binding molecule may be a halogenated nucleotide of the same type (e.g., BrdU), or a halogenated nucleotide of different types (e.g., BrdU and IdU). The nucleic acid binding molecule may be a halogenated nucleotide and a biotinylated nucleotide molecule. The nucleic acid binding molecule may be a biotinylated nucleotide molecule of different types (e.g., biotin-ATP, biotin-dGTP, biotin-dUTP, and / or biotin-dCTP).

[0079] Nucleic acid-binding molecules may be bound to nucleic acid ends by catalytic or non-catalytic means. Catalytic means may include non-enzymatic or enzymatic means. Nucleic acid-binding molecules may be bound to nucleic acid ends by an addition process using an enzymatic catalyst, for example, DNA polymerase I, terminal deoxynucleotidyl transferase (TdT), Klenow fragment, Phu polymerase, Taq polymerase, T4 DNA polymerase, T7 DNA polymerase, T4 polynucleotide kinase, or RNA polymerase. Non-catalytic means may include chemical factors, such as chemical factors having catalytic properties. Non-catalytic means may include physical or biochemical factors.

[0080] In embodiments where the nucleic acid terminus is a single-strand gap or single-strand nick, the nucleic acid binding molecule can be bound to the nucleic acid terminus by utilizing polymerase I. For example, in this embodiment, the nucleic acid binding molecule may be a biotinylated nucleotide molecule. In the context of this embodiment, the proteinase treatment is performed for approximately 30 seconds.

[0081] In embodiments where the nucleic acid terminus is a double-stranded blunt-end cleavage or a double-stranded 3'-overhang cleavage, the nucleic acid binding molecule may be bound to the nucleic acid terminus by utilizing TdT (or TdT reaction). For example, in this embodiment, the nucleic acid binding molecule may be a halogenated nucleotide or a nucleoside molecule (e.g., BrdU). In the context of this embodiment, the proteinase treatment is performed for approximately 60 seconds.

[0082] Those skilled in the art will understand that the conditions under which the nucleic acid binding molecule binds to the nucleic acid end may differ depending on the type of nucleic acid binding molecule. For example, if the nucleic acid binding molecule is a biotinylated nucleotide molecule, the conditions may require conditions optimal for the polymerase I reaction, such as a temperature of about 37°C. In this embodiment, the biological material may undergo a step to block endogenous biotin before the biotinylated nucleotide molecule is added. If the nucleic acid binding molecule is a halogenated nucleotide or nucleoside molecule (e.g., BrdU), the conditions may require conditions optimal for the TdT reaction, such as a temperature of about 37°C.

[0083] The methods described herein involve attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that is bound to the nucleic acid at the nucleic acid terminal (either directly or indirectly as described herein).

[0084] The two different binding molecules may each be an antibody or its fragment, a streptavidin molecule, an avidin molecule, a streptomycin analog, a biotin molecule, a peptide, a protein, a nucleic acid, an azide, or a polymer, or may contain any of these. The two different binding molecules may be of the same type or different types. For example, the two different binding molecules may be: (i) Different antibodies or fragments thereof, such as monoclonal antibodies (or their fragments) and polyclonal antibodies (or their fragments); (ii) Different antibodies or fragments thereof, such as monoclonal antibodies (or fragments thereof) that are specific to nucleic acid-binding molecules used in the method, and monoclonal antibodies (or fragments thereof) that are specific to nucleic acid-binding proteins (i.e., endogenous nucleic acid-binding proteins); (iii) Antibodies or fragments thereof, and streptavidin; (iv) Antibodies or fragments thereof, and avidin; (v) Antibodies or fragments thereof, and proteins or peptides; or (vi) Antibodies or their fragments, and biotin molecules It may be, or it may include them.

[0085] Preferably, the two different binding molecules are (or include) two different monoclonal antibodies (or their fragments); one is specific to the nucleic acid binding molecule used in the method, and the other is specific to the nucleic acid binding protein (i.e., an endogenous nucleic acid binding protein). Preferably, the two different binding molecules are (or include) two different monoclonal antibodies (or their fragments) derived from different species (e.g., rabbit and mouse).

[0086] The two different binding molecules may also attach to the oligonucleotide molecule. For example, each of the two different binding molecules may attach to the oligonucleotide molecule. Preferably, the oligonucleotide molecules of the two different binding molecules are different (e.g., have different sequences). Preferably, the two different binding molecules attach to different oligonucleotide molecules. Each oligonucleotide molecule may be covalently bonded to each of the two different binding molecules.

[0087] As used herein in the context of two different binding molecules, the term “different” means that each of the two binding molecules binds to a different target molecule than the other two different binding molecules. Specifically, in the methods, kits, and uses described herein, one set of two different binding molecules binds to a nucleic acid binding molecule, and the other set of two different binding molecules binds to a nucleic acid binding protein. The nucleic acid binding protein and the nucleic acid binding molecule are not the same molecule. The two different binding molecules may be molecules of the same type that have specificity for different molecules, for example, a monoclonal antibody (e.g., a mouse monoclonal antibody). Alternatively, the two different binding molecules may be molecules of different types, for example, a monoclonal antibody and a biotin molecule. Preferably, the two different binding molecules are monoclonal antibodies.

[0088] The two different binding molecules may include molecular probes. These molecular probes may be fluorescent agents and / or dyes. The two different binding molecules may include components of a fluorescence-activated protein (FAP)-dye activation by proximal anchoring (DAPA) system (Carpenter et al., 2020, "Protein Proximity Observed Using Fluorogen Activating Protein and Dye Activated by Proximal Anchoring (FAP-DAPA) System"). For example, one of the two different binding molecules may include FAP, and the other may include a HaloTag receptor. The two different binding molecules may further include antibodies that specifically bind to either a nucleic acid-binding molecule or a nucleic acid-binding protein. Thus, for example, one of the different binding molecules may be a fusion protein of a HaloTag receptor and an antibody against a nucleic acid-binding protein, and the other binding molecule may be a fusion protein of FAP and an antibody against a nucleic acid-binding molecule. The two different binding molecules may contain two complementary fragments of the split YFP Venus protein (Harmon et al., 2017, "A Bi-fluorescence complementation system to detect associations between the Endoplasmic reticulum and mitochondria"). The two different binding molecules may further contain antibodies that specifically bind to either the nucleic acid-binding molecule or the nucleic acid-binding protein. Thus, for example, one of the different binding molecules may be a fusion protein of the N-terminus of the Venus protein and an antibody against the nucleic acid-binding protein, and the other different binding molecule may be a fusion protein of the C-terminus of the Venus protein and an antibody against the nucleic acid-binding molecule. Each of the two different binding molecules may contain nanobodies, aptamers, or antibodies attached to one of the two oligonucleotide molecules (i.e., one of the two different binding molecules may be attached to one of the two oligonucleotide molecules, and the other of the two different binding molecules may be attached to the other oligonucleotide molecule).The two oligonucleotide molecules may function as templates for branched DNA signal amplification in a branched proximity hybridization assay. The two different binding molecules may each contain a donor and acceptor FRET probe.

[0089] Those skilled in the art will understand that, depending on the types of the two different binding molecules, the conditions under which the two different binding molecules bind to the nucleic acid binding molecule and the nucleic acid binding protein may differ. For example, if the two different binding molecules are monoclonal antibodies, the two different binding molecules may be incubated with the biological material for at least 30 minutes, preferably 60 minutes. The two different binding molecules may be incubated with the biological material at a temperature of 15°C to 30°C, preferably 20°C to 25°C.

[0090] The process of adding two different binding molecules to a biological material may include adding the two different binding molecules separately or together. It is preferable to add the two different binding molecules separately. Thus, one of the two different binding molecules may be added first, and the other different binding molecule may be added second. Preferably, the binding molecule that binds to a nucleic acid-binding protein (i.e., an endogenous nucleic acid-binding protein) is added first, and the binding molecule that binds to the nucleic acid-binding molecule is added second.

[0091] The process of adding two different binding molecules to a biological material involves the following steps: i. The process of adding two different binding molecules to a biological material; ii. The step of incubating one of two different binding molecules with a biological material for at least 30 minutes, preferably 60 minutes; iii. A step of optionally performing a washing step to remove any unbound molecules; iv. The process of adding two different other binding molecules to a biological material; v. The step of incubating two different other binding molecules together with a biological material for at least 30 minutes, preferably 60 minutes; and vi. A step in which a washing process is optionally performed to remove any unbound molecules. It may include.

[0092] The process of adding two different binding molecules to a biological material involves the following steps: i. The process of attaching two different binding molecules to a biological material (where the different binding molecules are specific to nucleic acid-binding proteins (i.e., endogenous nucleic acid-binding proteins)); ii. The step of incubating one of two different binding molecules with a biological material for at least 30 minutes, preferably 60 minutes; iii. A step of optionally performing a washing step to remove any unbound molecules; iv. The process of adding two different binding molecules to a biological material (where the different binding molecules are specific to the nucleic acid binding molecule); v. The step of incubating two different other binding molecules together with a biological material for at least 30 minutes, preferably 60 minutes; and vi. A step in which a washing process is optionally performed to remove any unbound molecules. It may include.

[0093] The methods described herein depend on the presence of nucleic acid-binding proteins within the biological material. Thus, the methods described herein depend on the detection of endogenous nucleic acid-binding proteins bound to nucleic acids at their termini. More specifically, the methods described herein depend on the detection of colocalization between endogenous nucleic acid-binding proteins and nucleic acid termini within the biological material.

[0094] As will be further described herein, nucleic acid-binding proteins can bind to nucleic acids directly or indirectly at their termini. Those skilled in the art will understand that the detection methods described herein depend on the presence of nucleic acid-binding proteins bound to nucleic acids at their termini. For detection to function, the nucleic acid-binding protein must be in close proximity to the nucleic acid at its termini so that detection of colocalization between the nucleic acid-binding protein and the nucleic acid termini can occur via the binding of two different binding molecules.

[0095] Nucleic acid-binding proteins may indirectly bind to nucleic acids at their termini through interactions with adapter proteins. Alternatively, they may indirectly bind to nucleic acids at their termini as part of a protein complex. For example, the XRCC1 protein indirectly binds to nucleic acids through interactions with PARP1. For example, RAD51 and RPA bind directly to nucleic acids.

[0096] Thus, since nucleic acid-binding proteins should have the ability to interact with or bind to nucleic acids (either directly or indirectly), nucleic acid-binding proteins may also be nucleic acid repair proteins. The nucleic acid-binding protein may also be a protein involved in repairing nucleic acid cleavage. Preferably, the nucleic acid-binding protein binds to the nucleic acid ends (i.e., nucleic acid cleavage) within the biological material.

[0097] The nucleic acid-binding protein may be a protein involved in homologous recombination of nucleic acids (e.g., DNA) during double-strand break repair. Thus, the nucleic acid-binding protein may be RAD51. The nucleic acid-binding protein may be a protein involved in the response to nucleic acid (e.g., DNA) damage. Therefore, the nucleic acid-binding protein may be replication protein A (RPA) (e.g., RPA70). The nucleic acid-binding protein may be, for example, a protein introduced during DNA replication that is involved in repair errors in nucleic acids (e.g., DNA). Thus, the nucleic acid-binding protein may be PMS2, PMS1, or MLH1.

[0098] Nucleic acid-binding proteins include p53, MSH2, ataxia telangiectasia and Rad3-related proteins, ATM serine / threonine kinase, RAD52, XRCC1, proliferating cell nuclear antigen, XPC, Ku70, Ku80, nibrin, DDB2, Bloom syndrome protein, CHEK2, RAD51C, DNA polymerase eta, Rad50, DDB1, RBBP8, FANCB, PALB2, H2AX, DNA repair and recombinant protein RAD54-like, PrimPol, REV1, terminal deoxynucleotidyl transferase, DNA polymerase NEU, Fanconi anemia, complementation group C, FANCF, ERCC8, Artemis, ubiquitin ligase, RNF4, TP53BP1, AP endonuclease, ERCC4, and transcription factor II. H, XRCC3, XRCC2, RecA, ERCC6, SLX4, sirtuin 1, PTEN, replication protein A2, replication protein A3, Alkb homolog 3, alpha-ketoglutarate-dependent dioxygenase, exonuclease 5, DNA polymerase alpha catalytic subunit, cyclin H, or PARP1 / 2 may also be present.

[0099] Nucleic acid-binding proteins may contain at least one post-translational modification. The post-translational modification may be amino acid phosphorylation, amino acid acetylation, amino acid glycosylation, or amino acid methylation. For example, the nucleic acid-binding protein may be a phosphorylated form of H2AX (also known as yH2AX).

[0100] Nucleic acid-binding proteins may also be nucleic acid (e.g., DNA) repair proteins. Nucleic acid-binding proteins include ATM, ATR, RPA, RAD51, MRE11, RAD17, RAD9A, RAD1, HUS1, TOPBP1, SMUG1, OGG1, PARP1, PARP2, PARP3, PARP, MGMT, TDP1, TDP2, MSH2, MSH3, MSH6, MLH1, PMS2, MSH4, MSH5, MSH3, PMS1, XPC, XPA, DDB1, DDB2, TFIIH, ERCC3, ERCC2, XRCC1, ERCC1, LIG1, RAD51B, RA D51D, HELQ, RAD52, BRCA1, SHLD1, SHLD2, MUS81, FANCA, FANCB, FANCC, LIG4, XRCC5, XRCC6, Ku70, Ku80, DNPH1, POLA, POLB, POLD, POLE, REV3L, POLQ, FEN1, TREX1, TREX2, EXO1, APTX1, HERC2, RNF8, RNF4, H2AX, BLM, WRN, RECQL4, ATRIP, PCNA, TP53, RIF1, TOPBP2 may also be used.

[0101] The nucleic acid-binding protein may also be a protein involved in replication. The nucleic acid-binding protein may be PCNA, RFC, RFA, Topo I, Topo II, polymerase a, or polymerase d.

[0102] Nucleic acid-binding proteins include genes: UNG, SMUG1, MBD4, TDG, OGG1, MUTYH (MYH), NTHL1 (NTH1), MPG, NEIL1, NEIL2, NEIL3, APEX1 (APE1), APEX2, LIG3, XRCC1, PNKP, APLF, HMCES, PARP1 (ADPRT), PARP2 (ADPRTL2), PARP3 (ADPRTL3), PARG, PARPBP, MGMT, ALKBH2 (ABH2), ALKBH3 (DEPC1), TDP1, TDP2 (TTRAP), SPRTN (Spartan), MSH2, MSH3, MSH6, MLH1, PMS2, MSH4, MSH5, MLH3, PMS1, PMS2P3 (PMS2L3), HFM1, XPC, RAD23B, CETN2, RAD23A, XPA, DDB1, DDB2 (XPE), RPA1, RPA2, RPA3, TFIIH, ERCC3 (XPB), ERCC2 (XPD), GTF2H1, GTF2H2, GTF2H3, GTF2H4, GTF2H5 (TTDA), GTF2E2, CDK7, CCNH, MNAT1, ERCC5 (XPG), ERCC1, ERCC4 (XPF), LIG1, ERCC8 (CSA), ERCC6 (CSB), UVSSA (KIAA1530), XAB2 (HCNP), MMS19, RAD51, RAD51B, RAD51D, HELQ (HEL308), SWI5, SWSAP1, ZSWIM7 (SWS1), SPIDR, PDS5B, DMC1, XRCC2, XRCC3, RAD52, RAD54L, RAD54B, BRCA1, BARD1, ABRAXAS1, PAXIP1 (PTIP), SMC5, SMC6, SHLD1, SHLD (FAM35A), SHLD3, SEM1 (SHFM1) (DSS1), RAD50, MRE11A, NBN (NBS1), RBBP8 (CtIP), MUS81, EME1 (MMS4L), EME2, SLX1A (GIYD1), SLX1B (GIYD2), GEN1, FANCA, FANCB, FANCC, BRCA2 (FANCD1), FANCD2, FANCE, FANCF, FANCG (XRCC9), FANCI (KIAA1794), BRIP1 (FANCJ), FANCL, FANCM, PALB2 (FANCN), RAD51C (FANCO), SLX4 (FANCP), FAAP20 (C1orf86),FAAP24 (C19 or F40), FAAP100, UBE2T (FANCT), XRCC6 (Ku70), XRCC5 (Ku80), PRKDC, LIG4, XRCC4, DCLRE1C (Artemis), NHEJ1 (XLF, Cernunnos), NUDT1 (MTH1), DUT, RRM2B (p53R2), PARK7 (DJ-1), DNPH1, NUDT15 (MTH2 ), NUDT18(MTH3), POLA1, POLB, POLD1, POLD2, POLD3, POLD4, POLE(POLE1), POLE2, POLE3, POLE4, REV3L (POLZ), MAD2L2(REV7), REV1(REV1L), POLG, POLH, POLI(RAD30B), POLQ, POLK(DINB1), POLL, POLM, POLN (POL4P), PRIMPOL, DNTT, FEN1 (DNaseIV), FAN1 (MTMR15), TREX1, TREX2, EXO1 (HEX1), APTX (aprataxin), SPO11, ENDOV, DNA2, DCLRE1A (SNM1A), DCLRE1B (SNM1B), EXO5, UBE2A (RAD6A) UBE2B (RAD6B), RAD18, SHPRH, HLTF(SMARCA3), RNF168, RNF8, RNF4, UBE2V2(MMS2), UBE2N(UBC13), USP1, WDR4 8, HERC2, H2AX (H2AFX), CHAF1A (CAF1), SETMAR (METNASE), ATRX, BLM, RMI1, TOP3A, WRN, RECQL4, ATM, MPL These may be DNA repair-related factors expressed by KIP(TTDN1), RPA4, PRPF19(PSO4), RECQL(RECQ1), ATR, ATRIP, MDC1, PCNA, RAD1, RAD9A, HUS1, RAD17(RAD24), CHEK1, CHEK2, TP53, TP53BP1(53BP1), RIF1, TOPBP1, CLK2, or PER1.

[0103] In embodiments where the nucleic acid terminus is a single-strand gap or single-strand nick, the nucleic acid binding molecule can bind to the nucleic acid terminus via a polymerase I catalyst. For example, in this embodiment, the nucleic acid binding molecule may be a biotinylated nucleotide molecule, and thus the nucleic acid terminus is extended by the addition of a nucleotide molecule. In this embodiment, for example, the nucleic acid binding protein may be PMS2, PMS1, or MLH1. The two different binding molecules in this example may be an anti-PMS2 (or anti-PMS1 or anti-MLH1) monoclonal antibody and an anti-biotin monoclonal antibody. Thus, a method for detecting nucleic acid terminus in a biological material containing nucleic acids is: a) A step of incubating the biological material with proteinase for less than 60 seconds; b) A step of adding a biotinylation molecule and polymerase I to a biological material under conditions such that the biotinylation molecule binds to the nucleic acid ends within the biological material; c) Adding an anti-PMS2 (or anti-PMS1 or anti-MLH1) monoclonal antibody and an anti-biotin monoclonal antibody to a biological material under conditions such that the anti-biotin monoclonal antibody binds to a biotinylation molecule bound to the nucleic acid terminus, and the anti-PMS2 (or anti-PMS1 or anti-MLH1) monoclonal antibody binds to PMS2 (or PMS1 or MLH1) which (indirectly) binds to the nucleic acid at the nucleic acid terminus; and d) A step of detecting nucleic acid ends in a biological material by detecting the colocalization of PMS2 (or PMS1 or MLH1) and nucleic acid ends via the binding of two monoclonal antibodies; It may include.

[0104] In embodiments where the nucleic acid end is a double-stranded blunt-end cleavage or a double-stranded 3'-overhang cleavage, the nucleic acid binding molecule can bind to the nucleic acid end via a terminal deoxyribonucleotide transferase (TdT or TdT reaction) catalyst. For example, in this embodiment, the nucleic acid binding molecule may be a halogenated nucleotide or nucleoside molecule (e.g., BrdU), and thus the nucleic acid end is extended by the addition of a nucleotide molecule. In this embodiment, for example, the nucleic acid binding protein may be RAD51 or RPA (e.g., RPA70). In this example, the two different binding molecules may be an anti-RAD51 (or anti-RPA) monoclonal antibody and an anti-BrdU monoclonal antibody. Thus, a method for detecting nucleic acid ends in a nucleic acid-containing biological material is: a) A step of incubating the biological material with proteinase for less than 2 minutes; b) Adding BrdU and TdT to a biological material under conditions such that BrdU binds to the nucleic acid ends within the biological material; c) Adding an anti-RAD51 (or anti-RPA) monoclonal antibody and an anti-BrdU monoclonal antibody to a biological material under conditions such that the anti-BrdU monoclonal antibody binds to BrdU bound to the nucleic acid terminal, and the anti-RAD51 (or anti-RPA) monoclonal antibody binds to RAD51 (or RPA) bound to the nucleic acid terminal; d) A step to detect nucleic acid ends in a biological material by detecting the colocalization of RAD51 (or RPA) and nucleic acid ends via the binding of two monoclonal antibodies. It may include.

[0105] The method may further include a step of adding a further binding molecule that binds to the two different binding molecules after the step of adding two different binding molecules. In this embodiment, the two different binding molecules do not have to be attached to the oligonucleotide molecule. In this embodiment, the further binding molecule may be attached to the oligonucleotide molecule, so that the further binding molecule links the oligonucleotide molecule to the two different binding molecules. Preferably, the further binding molecule is two different further binding molecules. In the method described herein, the two different further binding molecules may each be attached to a different oligonucleotide molecule (i.e., oligonucleotide molecules with different sequences).

[0106] Thus, a method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) A process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein attached to the nucleic acid terminal; d) A step of adding two different further binding molecules to the biological material (where one of the two different further binding molecules binds to the binding molecule bound to the nucleic acid binding molecule, and the other of the two different further binding molecules binds to the binding molecule bound to the nucleic acid binding protein); e) A step of detecting nucleic acid ends in biological material by detecting colocalization between nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules and two further binding molecules; It may include.

[0107] As used herein, the term “different” in the context of two further binding molecules means that each of the two further binding molecules binds to one of the binding molecules but not to the other. Thus, each of the further different molecules may be specific to a different binding molecule. The further binding molecules may be of the same type (e.g., monoclonal binding antibodies, each specific to a different binding molecule) or of different types. Preferably, the two different further binding molecules are monoclonal antibodies; one is an antibody that binds to a binding molecule bound to a nucleic acid binding molecule bound to a nucleic acid terminal, and the other is an antibody that binds to a binding molecule bound to a nucleic acid binding protein that binds to a nucleic acid at the nucleic acid terminal.

[0108] Further binding molecules may include antibodies or fragments thereof, streptavidin molecules, avidin molecules, biotin molecules, proteins, peptides, nucleic acids, azides, or polymers. Further binding molecules may consist of at least two distinct further binding molecules (e.g., two distinct further binding molecules specific to each of the two distinct binding molecules). For example, the two distinct binding molecules that bind to the nucleic acid binding molecule and the nucleic acid binding protein may be primary antibodies or fragments thereof, and the further binding molecules may be secondary antibodies or fragments thereof that specifically target the primary antibodies. For example, the two distinct binding molecules that bind to the nucleic acid binding molecule may be mouse and rabbit primary antibodies, and the further binding molecules may be anti-mouse and anti-rabbit secondary antibodies. Further binding molecules may be directly attached to (e.g., conjugated to) oligonucleotide molecules. Preferably, the two further binding molecules may each be directly attached to (e.g., conjugated to) different oligonucleotide molecules (i.e., oligonucleotide molecules with different sequences).

[0109] The method may further include, after the step of adding two different binding molecules (or after the step of adding further binding molecules), adding a further oligonucleotide molecule that hybridizes with the oligonucleotide molecule attached to the two different binding molecules (or further binding molecules) to form a cyclic amplification template.

[0110] Further oligonucleotide molecules may have the same sequence or at least two different sequences. Further oligonucleotide molecules may hybridize with two oligonucleotide molecules attached to two different binding molecules (or further binding molecules) to form a cyclic template. Two further oligonucleotide molecules may ligate together to form a cyclic template. Thus, the method may further include the step of ligating two further oligonucleotide molecules. The method may further include the steps of extending two further oligonucleotide molecules, followed by the steps of ligating them. Preferably, the further oligonucleotide molecules have at least two different sequences. The cyclic template may contain or be composed of two further oligonucleotide molecules. The cyclic template may further contain the extension products of two further oligonucleotide molecules. The cyclic template is suitable for rolling circle amplification. The cyclic template may be formed by ligating further oligonucleotide molecules (or two different further oligonucleotide molecules).

[0111] The method may further include a step of performing nucleic acid amplification and producing an amplification product before the step of detecting nucleic acid ends. Thus, detecting nucleic acid ends may include a step of performing nucleic acid amplification and producing an amplification product to be detected. The nucleic acid amplification may be rolling circle amplification.

[0112] Thus, a method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule attaches to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule attaches to a nucleic acid binding protein bound to the nucleic acid terminal (where the two different binding molecules each attach to an oligonucleotide molecule); d) A step of adding further oligonucleotide molecules to form a hybrid with oligonucleotide molecules attached to two different binding molecules to form a cyclic amplification template; e) A step of performing nucleic acid amplification operations to produce amplification products; and f) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0113] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule attaches to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule attaches to a nucleic acid binding protein bound to the nucleic acid terminal (where the two different binding molecules each attach to a different oligonucleotide molecule); d) A step of adding further oligonucleotide molecules to form a hybrid with oligonucleotide molecules attached to two different binding molecules to form a cyclic amplification template; e) A step of performing nucleic acid amplification operations to produce amplification products; and f) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0114] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule attaches to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule attaches to a nucleic acid binding protein bound to the nucleic acid terminal (where the two different binding molecules each attach to a different oligonucleotide molecule); d) A step of forming a cyclic amplification template by adding two further oligonucleotide molecules that hybridize with oligonucleotide molecules attached to two different binding molecules; e) A step of performing nucleic acid amplification operations to produce amplification products; and f) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0115] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 2 minutes using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule attaches to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule attaches to a nucleic acid binding protein bound to the nucleic acid terminal (where the two different binding molecules each attach to an oligonucleotide molecule); d) A step of adding further oligonucleotide molecules to form a hybrid with oligonucleotide molecules attached to two different binding molecules to form a cyclic amplification template; e) A step of performing nucleic acid amplification operations to produce amplification products; and f) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0116] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 2 minutes using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule attaches to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule attaches to a nucleic acid binding protein bound to the nucleic acid terminal (where the two different binding molecules each attach to a different oligonucleotide molecule); d) A step of adding further oligonucleotide molecules to form a hybrid with oligonucleotide molecules attached to two different binding molecules to form a cyclic amplification template; e) A step of performing nucleic acid amplification operations to produce amplification products; and f) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0117] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable in less than 2 minutes at a temperature of approximately 22°C using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule attaches to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule attaches to a nucleic acid binding protein bound to the nucleic acid terminal (where the two different binding molecules each attach to a different oligonucleotide molecule); d) A step of forming a cyclic amplification template by adding two further oligonucleotide molecules that hybridize with oligonucleotide molecules attached to two different binding molecules; e) A step of performing nucleic acid amplification operations to produce amplification products; and f) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0118] Thus, a method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) A process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein attached to the nucleic acid terminal; d) A step of attaching two different further binding molecules to the biological material (where one of the two different further binding molecules attaches to the binding molecule bound to the nucleic acid binding molecule, and the other of the two different further binding molecules attaches to the binding molecule bound to the nucleic acid binding protein, where each of the two different further binding molecules attaches to the oligonucleotide molecule); e) A step of forming a cyclic amplification template by adding further oligonucleotide molecules to hybridize with oligonucleotide molecules attached to two different further binding molecules; f) A process of performing nucleic acid amplification operations to produce amplification products; and g) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0119] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) A process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein attached to the nucleic acid terminal; d) A step of attaching two different further binding molecules to the biological material (where one of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding molecule, and the other of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding protein, where each of the two different further binding molecules attaches to a different oligonucleotide molecule); e) A step of forming a cyclic amplification template by adding further oligonucleotide molecules to hybridize with oligonucleotide molecules attached to two different further binding molecules; f) A process of performing nucleic acid amplification operations to produce amplification products; and g) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0120] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) A process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein attached to the nucleic acid terminal; d) A step of attaching two different further binding molecules to the biological material (where one of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding molecule, and the other of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding protein, where each of the two different further binding molecules attaches to an oligonucleotide molecule); e) A step of forming a cyclic amplification template by adding two different further oligonucleotide molecules to hybridize with oligonucleotide molecules attached to two different further binding molecules; f) A process of performing nucleic acid amplification operations to produce amplification products; and g) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0121] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 2 minutes using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) A process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein attached to the nucleic acid terminal; d) A step of attaching two different further binding molecules to the biological material (where one of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding molecule, and the other of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding protein, where each of the two different further binding molecules attaches to an oligonucleotide molecule); e) A step of forming a cyclic amplification template by adding further oligonucleotide molecules to hybridize with oligonucleotide molecules attached to two different further binding molecules; f) A process of performing nucleic acid amplification operations to produce amplification products; and g) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0122] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A process of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable in less than 2 minutes at a temperature of approximately 22°C using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) A process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein attached to the nucleic acid terminal; d) A step of attaching two different further binding molecules to the biological material (where one of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding molecule, and the other of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding protein, where each of the two different further binding molecules attaches to a different oligonucleotide molecule); e) A step of forming a cyclic amplification template by adding further oligonucleotide molecules to hybridize with oligonucleotide molecules attached to two different further binding molecules; f) A process of performing nucleic acid amplification operations to produce amplification products; and g) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0123] A method for detecting nucleic acid ends in a biological material containing nucleic acids is: a) A step of incubating a biological material with proteinase and increasing the accessibility to the nucleic acid ends to a level achievable at a temperature of approximately 22°C (i.e., room temperature) in less than 2 minutes using proteinase K at a concentration of 6 μg / mL to 8 μg / mL (e.g., approximately 7 μg / mL); b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) A process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein attached to the nucleic acid terminal; d) A step of attaching two different further binding molecules to the biological material (where one of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding molecule, and the other of the two different further binding molecules attaches to a binding molecule bound to a nucleic acid binding protein, where each of the two different further binding molecules attaches to a different oligonucleotide molecule); e) A step of forming a cyclic amplification template by adding two different further oligonucleotide molecules to hybridize with oligonucleotide molecules attached to two different further binding molecules; f) A process of performing nucleic acid amplification operations to produce amplification products; and g) A step to detect amplification products and detect nucleic acid ends within the biological material. It may include.

[0124] In the methods described herein, the detection of co-localization between a nucleic acid-binding protein and a nucleic acid terminus may include detection of an amplification product, a proximity ligation assay, or a proximity-driven reaction with a phosphor or dye. The detection of co-localization between a nucleic acid-binding protein and a nucleic acid terminus may also include detection of a branched probe or a FRET probe.

[0125] The process of detecting nucleic acid ends within biological material (e.g., detection of amplification products) may be carried out using microscopy, automated high-cell-number analysis methods, spectroscopy, fluorescence microscopy (broad-field, confocal, multifocal, super-resolution, catapulting microscopy, laser scanning, high-throughput, high-content), fluorescence measurement, transmitted light microscopy for absorbance detection, flow cytometry, cell sorting (FACS), and / or mass spectrometry. The process of detecting nucleic acid ends within biological material (e.g., detection of amplification products) may include attaching a labeling molecule that has the ability to recognize and bind to a detection molecule, such as an amplification product, or two binding molecules or two further binding molecules. For example, the labeling molecule may bind to a further oligonucleotide molecular sequence. The labeling molecule may contain nucleic acids. The labeling molecule may contain barcodes or tags.

[0126] The co-localization of nucleic acid-binding proteins with nucleic acid ends may be detected by photoactivation of the binding molecules described herein or by photoactivation of probes attached to the amplification product. The co-localization of nucleic acid-binding proteins with nucleic acid ends may also be detected by immunocytochemical analysis using antibodies against GFP that recognize both fragments of the split protein (e.g., polyclonal antiserum). The co-localization of nucleic acid-binding proteins with nucleic acid ends may be detected by fluorescence analysis of an activating phosphor or by fluorescence analysis of a probe or tag attached to the amplification product. The co-localization of nucleic acid-binding proteins with nucleic acid ends may also be detected by detection of the amplification product (e.g., by using a tag such as a fluorescent tag). The co-localization of nucleic acid-binding proteins with nucleic acid ends may also be detected by proximity-driven SNAr reactions of lysine-linked phosphors (Hymel et al., 2014, "Detection of Protein-Protein Interactions by Proximity-Driven SNAr Reactions of Lysine-Linked Fluorophores"). The co-localization of nucleic acid-binding proteins and nucleic acid ends may be detected by FRET probe detection. Thus, one of two different binding molecules (i.e., a donor probe) can be excited by an external light source and transfer its energy to the other different binding molecule (i.e., an acceptor probe). The excited acceptor probe can emit light of a different wavelength (usually a long wavelength), which can be detected and measured. The co-localization of nucleic acid-binding proteins and nucleic acid ends can be detected by a branched proximity hybridization assay, which can detect branched probes (e.g., DNA probes). Detection by the method described herein is preferably performed in situ.

[0127] The methods described herein may include one or more further steps prior to the step of incubating the biological material with a proteinase. The methods may include a step of permeabilizing the biological material (e.g., cells). The methods may include a further step of increasing accessibility to nucleic acid ends. The methods may include a step of blocking nonspecific binding sites for at least one molecule used in the methods. The methods may include a step of amplifying nucleic acid (DNA) ends within the biological material. The permeabilizing step of the biological material (e.g., cells) may include chemical and / or physical manipulations that impair the integrity of the biological material (e.g., cells) to enable or facilitate access of molecules used in the methods to nucleic acids and nucleic acid-binding proteins within the biological material (e.g., cells).

[0128] Further steps to increase accessibility to nucleic acid ends may be performed by the method described herein, in addition to the step of incubating the biological material with a proteinase. Further steps to increase accessibility to nucleic acid ends can further facilitate access of the molecules used in the method to nucleic acid ends within the biological material (e.g., cells).

[0129] The step of blocking nonspecific binding sites for at least one molecule used in the method may include blocking all nonspecific binding sites of the molecule used in the method. The term "block" means an operation that prevents the important molecule used in this assay, such as an antibody, from binding to targets other than the molecular target for which the selected molecule exhibits useful affinity in the assay. A typical example of blockade is the operation used in antibody detection (immunodetection) of a target molecule in a biological material. Antibodies are directed antagonistically to clearly defined antigens but can also bind to various cellular elements through weak chemical interactions. This nonspecific binding can be minimized by pre-adding a blocking agent (such as albumin) that occupies such binding sites. Thus, the method described herein may further include a step of blocking nonspecific binding sites of molecules after the steps of adding two different binding molecules to the biological material and / or adding further binding molecules.

[0130] The step of modifying the nucleic acid (DNA) ends within the biological material may be performed after the proteinase treatment step and before the step of adding nucleic acid-binding molecules. The nucleic acid end modification step may be performed by chemical or physical means. For example, nucleic acid end modification may be performed by T4 polynucleotide kinase.

[0131] The methods described herein may include one or more washing steps. For example, proteinase and / or proteinase inhibitors may be washed off after the step of incubating the biological material with proteinase. The washing step may be performed after the step of adding nucleic acid binding molecules. The washing step may be performed after the step of adding two different binding molecules. The washing step may be performed after the step of adding further oligonucleotide molecules. The washing step may be performed after the step of performing amplification. The washing step removes unbound material, which may generate a clear detection signal and / or reduce background noise.

[0132] Figure 1 shows an exemplary method of the present invention after the proteinase treatment step. In step 1, a single-stranded nucleic acid (e.g., DNA) cleavage occurs. An endogenous nucleic acid-binding protein (e.g., PMS2, PMS1, or MLH1) binds to the nucleic acid at the nucleic acid end, for example, to fix the cleavage that occurred. In step 2, nucleic acid-binding molecules bind to the nucleic acid end. This can be achieved with the help of an enzyme, e.g., polymerase I (labeled by a gray circle in step 2). This forms a chain of nucleic acid-binding molecules (visualized in step 3). In step 3, two different binding molecules (attached to oligonucleotide molecules) bind to the nucleic acid-binding protein and nucleic acid-binding molecules, respectively. In step 4, further oligonucleotide molecules hybridize with the oligonucleotide molecules attached to the two different binding molecules. In step 5, further oligonucleotide molecules ligate to form a circular template. In step 6, rolling circle amplification is performed on the circular template to produce an amplification product. In step 7, a detection molecule (e.g., a fluorescent probe) binds to the amplification product, enabling detection of the amplification product.

[0133] Figure 2 shows yet another example of the method of the present invention. Here, the steps of the method are the same as those in Figure 1, except that an additional binding molecule (attached to the oligonucleotide molecule) binds to two different binding molecules (not attached to the oligonucleotide molecule in this example).

[0134] Figure 3 shows another example of the method of the present invention. The steps are the same as in Figure 1. However, here, the nucleic acid binding molecule (e.g., a nucleotide analog) is directly incorporated into the single-stranded nucleic acid (e.g., DNA) cleavage. Thus, in this example, no chain of nucleic acid binding molecules is formed.

[0135] Figure 4 shows another example of the method of the present invention. In this example, the nucleic acid cleavage is a double-strand nucleic acid cleavage. An endogenous nucleic acid-binding protein (e.g., RAD51 or RPA) binds to the nucleic acid at the nucleic acid end, for example, to fix the resulting cleavage. In step 2, the nucleic acid-binding molecule binds to the nucleic acid end. This can be achieved with the help of an enzyme, for example, TdT (labeled with a gray circle in step 2). This forms a chain of nucleic acid-binding molecules (visualized in step 3). In step 4, two different binding molecules bind to the nucleic acid-binding protein and the nucleic acid-binding molecule, respectively. In step 5 (optional), further binding molecules attached to the oligonucleotide molecule bind to two different binding molecules. In step 6, the further oligonucleotide molecule hybridizes with the oligonucleotide molecule attached to the further binding molecule, ligating to form a cyclic template. In step 7, rolling circle amplification is performed on the cyclic template to produce an amplification product. In step 8, a detection molecule (e.g., a fluorescent probe) binds to the amplification product, enabling detection of the amplification product.

[0136] The methods described herein enable the direct detection and visualization of nucleic acid ends. The methods described herein enable the labeling of the presence and location of single-strand nucleic acid breaks. Thus, the methods are particularly useful in detecting nucleic acid damage, which has broad applicability. For example, the methods may be used to evaluate the characteristics of DNA in a subject. The methods may also be used to study the mechanisms of action of DNA damage induction, transmission, and repair. From the perspective of cancer cells, the methods can be particularly useful in evaluating the efficacy of therapeutic agents.

[0137] Thus, in one embodiment, the present invention is a method for evaluating the effectiveness of a therapeutic agent, a) A step of performing the detection method described herein on a sample obtained from a subject before administering the therapeutic agent; b) A step of performing the detection method described herein on a sample obtained from a subject after administering a therapeutic agent; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where the therapeutic agent is effective if the amount of nucleic acid ends detected in step a) is greater than that detected in step b)). This includes, where steps a) and b) can be carried out in any order. Provide a method.

[0138] The subject may be an animal, but preferably a human. Preferably, the nucleic acid ends are DNA ends. More preferably, the nucleic acid ends are single-stranded nucleic acid (e.g., DNA) ends.

[0139] The therapeutic agent may be an oncological drug or a drug candidate. The method may be used to evaluate the therapeutic efficacy of a drug candidate. The therapeutic agent may target proteins involved in the mismatch repair (MMR) pathway. Thus, the therapeutic agent may target PMS1, PMS2, and / or MLH1. Therefore, in the detection method described herein, the nucleic acid-binding protein may be PMS1, PMS2, or MLH1. The therapeutic agent may inhibit proteins involved in the mismatch repair pathway. Thus, once inhibited, the protein will be unable to bind to nucleic acid ends, and therefore the detection method will show a small number of nucleic acid ends detected in the biological material.

[0140] Samples may be obtained from subjects at least 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after administration of the therapeutic agent. The method may include performing a nucleic acid end detection method at multiple time points after administration of the therapeutic agent. This solution can evaluate the long-term effects of the therapeutic agent.

[0141] In one embodiment, the present invention relates to a method for evaluating the effectiveness of a therapeutic agent, a) A step of performing the detection method described herein on a sample obtained from a subject before administering the therapeutic agent; b) A step of performing the detection method described herein on a sample obtained from a subject after administering a therapeutic agent; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where the therapeutic agent is effective if the amount of nucleic acid ends is greater in step b) than in step a). This includes, where steps a) and b) can be carried out in any order. Provide a method.

[0142] The subject may be an animal, but it is preferable that the subject be a human. Preferably, the nucleic acid end is a DNA end. More preferably, the nucleic acid end is a double-stranded nucleic acid (e.g., DNA) end.

[0143] The therapeutic agent may be an oncological drug or a candidate drug. The method may be used to evaluate the therapeutic effect of the candidate drug. The therapeutic agent may target a protein associated with the repair of errors in nucleic acids (e.g., DNA), or a protein associated with homologous recombination of nucleic acids (e.g., DNA) during double-strand break repair. Thus, the therapeutic agent may target RPA or Rad51. The therapeutic agent may inhibit DNA synthesis, cause nucleic acid damage, and / or inhibit DNA damage repair.

[0144] Samples may be obtained from subjects at least 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after administration of the therapeutic agent. The method may include performing a nucleic acid end detection method at multiple time points after administration of the therapeutic agent. This solution can evaluate the long-term effects of the therapeutic agent.

[0145] The sample may be obtained from a cancer biopsy. The sample may contain cancer cells. The methods described herein may also be useful for evaluating nucleic acid damage caused by drugs (e.g., chemicals or environmental substances).

[0146] Thus, in one embodiment, the present invention relates to a method for evaluating nucleic acid damage caused by a drug, a) A step of performing the detection method described herein on a sample obtained from a subject before administration of the drug or before exposure to the drug; b) The step of performing the detection method described herein on a sample obtained from a subject after administration of a therapeutic agent or exposure to a drug; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where the drug causes nucleic acid damage if the amount of nucleic acid ends is greater in step b) than in step a). This includes, where steps a) and b) can be carried out in any order. Provide a method.

[0147] Nucleic acid damage can be single-strand or double-strand nucleic acid breaks. Preferably, the nucleic acid end is a DNA end. Thus, nucleic acid damage may be single-strand DNA breaks or double-strand DNA breaks. In the case of single-strand breaks, the method preferably depends on the detection of PMS1, PMS2, or MLH1 as the nucleic acid binding protein. In the case of double-strand breaks, the method preferably depends on the detection of Rad51 or RPA as the nucleic acid binding protein. The drug may be a therapeutic agent, a chemical substance, or an environmental substance.

[0148] In the case of therapeutic agents, the methods described herein can detect nucleic acid damage caused by therapeutic agents that may have side effects. For example, a therapeutic agent may exhibit therapeutic effects against a particular disease or disorder, and at the same time may cause nucleic acid damage in a particular cell type. Thus, the methods described herein can identify whether a therapeutic agent causes nucleic acid (e.g., DNA) damage.

[0149] The chemical substance may be a toxin, ink, adhesive, paint, oil, lubricant, hair dye, laboratory reagent, welding fume, hazardous drug, or cleaning solution.

[0150] Environmental agents may be pollutants, bacteria (or parts thereof), viruses (or parts thereof), or radioactive materials. Pollutants may be air pollutants (e.g., particulate matter, ozone, nitrogen dioxide, carbon monoxide, or sulfur dioxide), water pollutants (e.g., bacteria, viruses, parasites, fertilizers, pesticides, pharmaceuticals, nitrates, phosphates, plastics, or fecal waste), or soil pollutants (e.g., pesticides, petroleum products, radon, asbestos, lead, chromified copper arsenate, or creosote).

[0151] As used herein, the term “administration” refers to any type of administration of a drug to a subject. For example, a drug may be administered to a subject by any acceptable route of administration, including but not limited to oral, topical (including transdermal), and parenteral modes of administration.

[0152] As used herein, the term “exposure to a drug” encompasses all types of exposure. For example, an object may be exposed to a drug by inhaling it from the air, consuming it, absorbing it through the skin, eyes, or membranes within the object’s body (e.g., nasal or vaginal mucosa), or (e.g., in the case of a wound) by fluid contact with the drug.

[0153] Samples may be obtained from subjects at least 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or 1 year after administration of the therapeutic agent or exposure to the therapeutic agent. The method may include performing a nucleic acid end detection method at multiple time points after administration of the therapeutic agent. This solution can evaluate the long-term effects of the therapeutic agent. The method may include performing a nucleic acid end detection method at multiple time points after administration of the drug or exposure to the drug. This solution can evaluate the long-term effects of the drug on the health status of subjects. The methods described herein are preferably performed in vitro.

[0154] The present invention provides a method for predicting the response of a subject diagnosed with cancer to anti-cancer treatment, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by performing the detection method described herein; and (ii) predicting the response of the subject to anti-cancer treatment based on the measured level of nucleic acid-binding proteins in the sample.

[0155] The present invention provides a method for selecting a customized treatment for a subject diagnosed with cancer, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by performing the detection method described herein; and (ii) selecting a customized treatment for the subject based on the measured level of nucleic acid-binding proteins in the sample.

[0156] The present invention provides a method for classifying subjects diagnosed with cancer into patient cohorts, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subjects by performing the detection method described herein; and (ii) classifying the subjects diagnosed with cancer into patient cohorts based on the measured level of nucleic acid-binding proteins in the sample.

[0157] The present invention provides a method for predicting whether a tumor from a subject diagnosed with cancer has the ability to repair DNA by homologous recombination, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by carrying out the detection method described herein; and (ii) predicting, based on the level of nucleic acid-binding proteins measured in the sample, whether the tumor derived from the subject diagnosed with cancer can be repaired by homologous recombination.

[0158] The present invention provides a method for evaluating the state of mismatch repair pathways in a sample containing tumor cells obtained from a subject, comprising: (i) measuring the level of nucleic acid-binding proteins in tumor cells by carrying out the detection method described herein; and (ii) evaluating the state of mismatch repair pathways based on the measured level of nucleic acid-binding proteins in tumor cells.

[0159] In all embodiments described herein, the nucleic acid-binding protein is preferably RAD51, RPA (e.g., RPA70), PMS1, PMS2, or MLH1. In all embodiments described herein, the sample preferably contains tumor cells.

[0160] Anticancer treatments or anticancer agents may include poly-ADP-ribose polymerase (PARP) inhibitors. PARP is a protein (enzyme) that helps repair damaged cells. As a cancer treatment, PARP inhibitors stop the implementation of this repair mechanism in cancer cells, causing them to die. Normally, the BRCA1 and BRCA2 genes play a certain role in cell repair in the body. If one or both of these genes are defective, cells are less likely to repair themselves. Patients with BRCA gene defects have an increased risk of certain cancers, including breast cancer, ovarian cancer, and prostate cancer. Cancer cells with BRCA gene defects already have a fragile repair system. Thus, blocking PARP with PARP inhibitors means that cells cannot repair themselves and die.

[0161] In all embodiments described herein, preferably, the subject has not been administered chemotherapy or anticancer agents at least 24 hours prior to the isolation of the sample. In all embodiments described herein, preferably, the sample has not been exposed to DNA inducers or the environment prior to the step of measuring the level of nucleic acid-binding proteins in the sample.

[0162] In all embodiments described herein, preferably, the step of measuring the level of nucleic acid-binding proteins in a sample includes detecting or visualizing the location and / or distribution of nucleic acid-binding proteins in the sample (e.g., cells such as tumor cells). The step of measuring the level of nucleic acid-binding proteins in a sample may include measuring the level of cells having nucleic acid-binding proteins in the sample. Preferably, the cells are tumor cells.

[0163] In all embodiments described herein, preferably, the measured level of nucleic acid-binding protein is compared to a control value. If the level of nucleic acid-binding protein in the sample is lower than the control value, this indicates that (i) the subject is expected to respond to anticancer treatment, (ii) the selected therapy contains an agent specific to treat tumors with impaired DNA damage response, (iii) the subject belongs to a cohort characterized by a response to anticancer treatment, or (iv) the tumor has DNA repair ability through homologous recombination. If the level of nucleic acid-binding protein in the sample is higher than the control value, this indicates that (i) the subject is expected not to respond to anticancer treatment, (ii) the selected therapy does not contain an agent specific to treat tumors with impaired DNA damage response, (iii) the subject belongs to a patient cohort characterized by a lack of response to anticancer treatment, or (iv) the tumor does not have DNA repair ability through homologous recombination.

[0164] The term "control value" refers to a laboratory value used as a standard for values / data obtained from samples taken from a subject. A control value may be based on values ​​obtained from a group of patients considered representative. Alternatively, a control value may be based on individual samples, such as values ​​obtained from patient-derived samples at a previous point in time. For example, a control value may be from samples taken from patients without specific mutations that affect responsiveness to anti-cancer treatment.

[0165] The term "tumor with impaired DNA damage response" refers to a tumor or tumor cells with altered DNA damage response pathways. This may include abnormal expression of proteins involved in the DNA damage response. These proteins include ATM, ATR, RPA, RAD51, MRE11, RAD17, RAD9A, RAD1, HUS1, TOPBP1, SMUG1, OGG1, PARP1, PARP2, PARP3, PARP, MGMT, TDP1, TDP2, MSH2, MSH3, MSH6, MLH1, PMS2, MSH4, MSH5, MSH3, PMS1, XPC, XPA, DDB1, DDB2, TFIIH, ERCC3, ERCC2, XRCC1, ERCC1, LIG1, RAD51B, and RAD5 1D, HELQ, RAD52, BRCA1, SHLD1, SHLD2, MUS81, FANCA, FANCB, FANCC, LIG4, XRCC5, XRCC6, Ku70, Ku80, DNPH1, POLA, POLB, POLD, POLE, REV3L, POLQ, FEN1, TREX1, TREX2, EXO1, APTX1, HERC2, RNF8, RNF4, H2AX, BLM, WRN, RECQL4, ATRIP, PCNA, TP53, RIF1, and TOPBP2 may also be included. Modification of the DNA damage response pathway may lead to genomic instability.

[0166] The state of the mismatch repair pathway may indicate that one or more proteins involved in mismatch repair are deficient or malfunctioning. Alternatively, the state of the mismatch repair pathway may indicate that tumor cells have a fully functioning mismatch repair pathway. The present invention also provides a drug for use in the treatment of cancer in a subject, wherein the subject is identified as a responder to the drug in one manner described herein.

[0167] The methods described herein can be performed using the kits described herein. Those skilled in the art will understand that all molecules, reagents, and approaches described in the context of the methods are also applicable to embodiments of the kits of the present invention. However, certain preferred embodiments are also highlighted in the context of embodiments of the kits of the present invention.

[0168] The present invention is a kit for detecting nucleic acid ends in biological materials, a) Nucleic acid binding molecules; b) A binding molecule that binds to the nucleic acid binding molecule; and c) Binding molecules that bind to nucleic acid-binding proteins We provide a kit that includes this.

[0169] The present invention also relates to the use of a kit for detecting nucleic acid ends in biological materials, wherein the kit is a) Nucleic acid binding molecules; b) A binding molecule that binds to the nucleic acid binding molecule; and c) Binding molecules that bind to nucleic acid-binding proteins It provides its use, including. The kit may also contain a proteinase, preferably proteinase K.

[0170] Kits for detecting nucleic acid ends in biological materials are a) Nucleic acid binding molecules; b) A monoclonal antibody that binds to the nucleic acid-binding molecule; and c) Monoclonal antibodies that bind to nucleic acid-binding proteins It may include.

[0171] Kits for detecting nucleic acid ends in biological materials are a) Proteinases (e.g., proteinase K); b) nucleic acid binding molecules; c) A binding molecule that binds to the nucleic acid binding molecule; d) Binding molecules that bind to nucleic acid-binding proteins; and e) Further binding molecules It may include.

[0172] Kits for detecting nucleic acid ends in biological materials are a) hydrophilic solid support; b) nucleic acid binding molecules; c) A binding molecule that binds to the nucleic acid binding molecule; and d) Binding molecules that bind to nucleic acid-binding proteins It may include.

[0173] Kits for detecting nucleic acid ends in biological materials are a) Nucleic acid binding molecules; b) A binding molecule that binds to the nucleic acid binding molecule; c) Binding molecules that bind to nucleic acid-binding proteins; d) Reagents for amplification It may include.

[0174] Kits for detecting nucleic acid ends in biological materials are a) Nucleic acid binding molecules; b) A binding molecule that binds to the nucleic acid binding molecule; c) Binding molecules that bind to nucleic acid-binding proteins; and d) Further oligonucleotide molecules It may include.

[0175] Preferably, the hydrophilic solid support is a positively charged solid support. For example, the hydrophilic solid support may be a TOMO® slide or a poly-L-lysine coated slide.

[0176] The nucleic acid binding molecule may include a halogenated nucleotide or nucleoside molecule, a DNA precursor analog, and / or a biotinylated nucleotide molecule. The kit may contain at least one type of nucleic acid binding molecule. The kit may contain at least two types, at least three types, or at least four types of nucleic acid binding molecules.

[0177] The binding molecule that binds to the nucleic acid binding molecule may be an antibody or its fragment, a streptavidin molecule, an avidin molecule, a streptomycin analog, a biotin molecule, a peptide, a protein, a nucleic acid, an azide, or a polymer.

[0178] Two different binding molecules may be of the same type of binding molecule or different types of binding molecules. For example, the two different binding molecules are (i) different antibodies or fragments thereof, such as monoclonal antibodies (or fragments thereof) and polyclonal antibodies (or fragments thereof); (ii) different antibodies or fragments thereof, such as a monoclonal antibody (or fragment thereof) specific for the first nucleic acid binding molecule used in the method, and a monoclonal antibody (or fragment thereof) specific for the second nucleic acid binding molecule used in the method; (iii) an antibody or fragment thereof, and streptavidin; (iv) an antibody or fragment thereof, and avidin; (v) an antibody or fragment thereof, and a protein or peptide; or (vi) an antibody or fragment thereof, and a biotin molecule may be.

[0179] Preferably, the two different binding molecules are (or include) two different monoclonal antibodies (or fragments thereof); one is specific for the nucleic acid binding molecule used in the method, and the other is specific for a nucleic acid binding protein (i.e., an endogenous nucleic acid binding protein). Preferably, the two different binding molecules are (or include) two different monoclonal antibodies (or fragments thereof) from different species (e.g., rabbit and mouse).

[0180] In one embodiment, the two different binding molecules may be attached to an oligonucleotide molecule. Preferably, each of the two different binding molecules is attached to an oligonucleotide molecule. The oligonucleotide molecules attached to each of the two different binding molecules are preferably different (i.e., have different sequences).

[0181] In yet another embodiment, the kit may further include additional binding molecules that attach to the oligonucleotide molecule. The additional binding molecules may be of the same type or different types. Preferably, the additional binding molecules may be of different types. For example, the additional binding molecules may be two (different) antibodies (or their fragments). The additional binding molecules may be specific and / or selective for binding to two different binding molecules.

[0182] Further oligonucleotide molecules can preferably form hybrids with binding molecules present in the kit or oligonucleotide molecules attached to further binding molecules. Further oligonucleotide molecules may include the same or different types of oligonucleotides (e.g., having the same or different sequences). Preferably, further oligonucleotide molecules include at least two types of oligonucleotides (i.e., at least two oligonucleotide sequences). Further binding molecules may ligate to form a cyclic template.

[0183] The reagents for amplification may include polymerase (e.g., phi29 polymerase), dNTP mix, primer (e.g., hexamer primer), and / or nuclease-free. The kit may include reagents for fixing biological materials (e.g., cells). For example, the reagent for fixing biological materials may be 70% (v / v) ethanol.

[0184] The kit may further contain polymerases and / or ligases. The kit may also contain DNA polymerase I, TdT, Krenoh fragments, Phu polymerase, Taq polymerase, T4 DNA polymerase, T7 DNA polymerase, T4 polynucleotide kinase, and / or RNA polymerase. The present invention provides the kit described herein for use in the manner described herein.

[0185] The details described above are provided for illustrative purposes and examples only and do not limit the scope of the appended claims. Many variations in preferred embodiments of the invention as shown herein will be obvious to those skilled in the art and remain within the scope of the appended claims and their equivalents.

[0186] The present invention is further disclosed in the following sections: 1. A method for detecting nucleic acid ends in a biological material containing nucleic acids, a) A process of incubating a biological material with a proteinase to increase accessibility to the nucleic acid ends within the biological material without significantly affecting the level of nucleic acid-binding proteins within the biological material; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule bound to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein bound to the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. Methods that include...

[0187] 2. A method for detecting nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating a biological material with a proteinase to increase the accessibility of nucleic acid ends to a concentration achieved by using a proteinase (e.g., proteinase K) at a concentration of 6 μg / mL to 8 μg / mL for less than 2 minutes; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. A method that may include this.

[0188] 3. A method for detecting nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating the biological material with proteinase for less than 2 minutes; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. Methods that include...

[0189] 4. The method described in any one of items 1-3, wherein the biological material is an animal, plant, protozoan, bacterium or virus, or derived therefrom. 5. The method described in any one of items 1-4, wherein the biological material comprises cells or fragments thereof. 6. The method described in any one of items 1-5, wherein the biological material includes fixed cells. 7. The method according to any one of items 1 to 6, wherein the nucleic acid is DNA and / or RNA, preferably DNA, or is one thereof. 8. The method according to any one of items 1 to 7, wherein the nucleic acid end is a DNA end or an RNA end, preferably a DNA end. 9. The method according to any one of items 1 to 8, wherein the nucleic acid end is a single-stranded nucleic acid break or a double-stranded nucleic acid break. 10. The method according to any one of items 1 to 9, wherein the nucleic acid end is a single-strand gap, a double-strand blunt end break, a double-strand 3'-overhang end break, or a single-strand nick.

[0190] 11. The method according to any one of items 1 to 10, wherein the proteinase is a broad-spectrum proteinase. 12. The method according to any one of items 1 to 11, wherein the proteinase is aspartate protease, glutamate protease, metalloprotease, cysteine ​​protease, serine protease, or threonine protease, preferably the protease is a serine protease such as proteinase K. 13. The method according to any one of items 1 to 12, wherein the step of incubating the biological material with proteinase is carried out for at least 5 seconds, preferably at least 10 seconds, and more preferably at least 15 seconds. 14. The method according to any one of items 1 to 13, wherein the step of incubating the biological material with proteinase is carried out over a period of 1 second to less than 2 minutes, 15 seconds to 90 seconds, 20 seconds to 80 seconds, 25 seconds to 70 seconds, or 30 seconds to 60 seconds, preferably the incubation of the biological material with proteinase is carried out over a period of 30 seconds to 60 seconds. 15. The method according to any one of items 1 to 14, wherein the step of incubating the biological material with proteinase is carried out over a period of less than 100 seconds, less than 90 seconds, less than 80 seconds, less than 70 seconds, or less than 60 seconds, preferably less than 90 seconds.

[0191] 16. The method according to any one of items 1-15, wherein the step of incubating the biological material with proteinase is performed for approximately 30 seconds or approximately 60 seconds. 17. The method according to any one of items 1 to 16, wherein the step of incubating a biological material with a proteinase is terminated by the addition of a proteinase inhibitor. 18. The method according to any one of items 1 to 17, wherein the step of incubating the biological material together with the protease is carried out at a temperature of 15°C to 30°C, preferably 20°C to 25°C. 19. The method according to any one of items 1 to 18, wherein the protease is in solution, preferably present at a concentration of 1 μg / mL to 20 μg / mL, 3 μg / mL to 15 μg / mL, 5 μg / mL to 10 μg / mL, 6 μg / mL to 8 μg / mL, preferably at a concentration of about 7 μg / mL. 20. The method according to item 19, wherein the solution contains a buffer, urea and / or SDS.

[0192] 21. The nucleic acid binding molecule is (i) a halogenated nucleotide or nucleoside molecule such as BrdU, IdU, CldU; (ii) a DNA precursor analog such as EdU (5-ethynyl-2'-deoxyuridine), F-ara-EdU, 5-ethynyl-2'-deoxycytidine; [[ID=1%]]](iii) a biotinylated nucleotide molecule; (iv) an ADP-ribose molecule; (v) a protein molecule; (vi) a nucleotide or nucleoside molecule labeled with a label (where optionally, the label is selected from the group consisting of a fluorescent molecule, or a chemiluminescent molecule, or a radioisotope, or an enzyme substrate, or a biotin molecule) The method according to any one of items 1 to 20, which is selected from the group consisting of.

[0193] 22. The method according to any one of items 1 to 21, wherein the nucleic acid binding molecule is a halogenated nucleotide or nucleoside molecule, a DNA precursor analog, and / or a biotinylated nucleotide molecule. 23. The method according to any one of items 1 to 22, wherein a nucleic acid binding molecule binds to the ends of nucleic acids in a biological material by an addition process catalyzed with an enzyme catalyst using, for example, DNA polymerase I, TdT, Klenow fragment, Phu polymerase, Taq polymerase, T4 DNA polymerase, T7 DNA polymerase, T4 polynucleotide kinase, or RNA polymerase. 24. The method according to any one of items 1 to 23, wherein the two different binding molecules are two different monoclonal antibodies (or their fragments), or comprise them. 25. The method according to any one of items 1 to 24, wherein two different binding molecules are incubated with a biological material for at least 30 minutes, preferably 60 minutes.

[0194] 26. The method according to any one of items 1 to 27, wherein two different binding molecules are incubated with a biological material at a temperature of 15°C to 30°C, preferably 20°C to 25°C. 27. The method according to any one of items 1 to 26, wherein the step of adding two different binding molecules to a biological material may include adding the two different binding molecules separately or together, preferably the two different binding molecules are added separately, preferably one of the two different binding molecules is added first and the other different binding molecule is added subsequently.

[0195] 28. The process of adding two different binding molecules to a biological material is a. The step of adding two different binding molecules to the biological material; b. The step of incubating two different binding molecules together with a biological material for at least 30 minutes, preferably 60 minutes; c. A wash step may be optionally performed to remove any unbound or bound molecules; d. The process of adding two different other binding molecules to a biological material; e. A step of incubating two different other binding molecules together with the biological material for at least 30 minutes, preferably 60 minutes; f. A step in which a washing step may be optionally performed to remove any unbound or bound molecules. The method described in any one of items 1 through 27, including the method described in item 1.

[0196] 29. The method according to any one of items 1 to 28, wherein the nucleic acid-binding protein is a protein found in biological material. 30. The method according to any one of items 1 to 29, wherein a nucleic acid-binding protein binds to a nucleic acid directly or indirectly at the nucleic acid terminus. 31. The method according to item 30, wherein a nucleic acid-binding protein indirectly binds to a nucleic acid at its terminus via interaction with an adapter protein. 32. The method according to any one of items 1 to 31, wherein the nucleic acid-binding protein is a nucleic acid repair protein, preferably a nucleic acid end (e.g., cleavage) repair protein.

[0197] 33. Nucleic acid-binding proteins include RAD51, RPA (e.g., RPA70), PMS2, MLH1, PMS1, p53, MSH2, ataxia telangiectasia and Rad3-related proteins, ATM serine / threonine kinase, RAD52, XRCC1, proliferating cell nuclear antigen, XPC, Ku70, Ku80, nibrin, DDB2, Bloom syndrome protein, CHEK2, RAD51C, DNA polymerase eta, Rad50, DDB1, RBBP8, FANCB, PALB2, H2AX, yH2AX DNA repair and recombinant protein RAD54-like, Primpol, REV1, terminal deoxynucleotidyl transferase, DNA polymerase neu, Fanconi anemia, complementation group C, FANCF, ERCC8, Artemis, ubiquitin ligase, RNF4, TP53BP1, AP endonuclease, ERCC4, transcription factor II The method according to any one of items 1 to 32, wherein H, XRCC3, XRCC2, RecA, ERCC6, SLX4, sirtuin 1, PTEN, replication protein A2, replication protein A3, Alkb homolog 3, alpha-ketoglutarate-dependent dioxygenase, exonuclease 5, DNA polymerase alpha catalytic subunit, cyclin H, PARP1 / 2.

[0198] 34. The method according to any one of items 1 to 33, wherein the nucleic acid-binding protein includes post-translational modifications. 35. The method according to any one of items 1 to 34, wherein two different binding molecules each attach to an oligonucleotide molecule. 36. The method according to any one of items 1 to 35, wherein two different binding molecules each attach to a different oligonucleotide molecule. 37. The method according to any one of items 1 to 36, further comprising the step of adding two different binding molecules, followed by the step of adding further binding molecules, each of which binds to two different binding molecules. 38. The method of item 37, wherein the further binding molecules are two different further binding molecules. 39. The method according to item 37 or 38, wherein further binding molecules attach to the oligonucleotide molecule. 40. The method according to any one of items 37-39, wherein two different additional binding molecules each attach to a different oligonucleotide molecule.

[0199] 41. The method according to any one of items 1 to 40, further comprising the step of adding a further oligonucleotide molecule to the two different binding molecules (or to the further binding molecules, depending on items 37 to 40) to hybridize with the oligonucleotide molecule attached to the two different binding molecules (or to the further binding molecule) to form a cyclic template. 42. The method according to any one of items 1 to 41, further comprising the step of adding two different further oligonucleotide molecules to the two different binding molecules (or further binding molecules) after the step of adding two different binding molecules (or further binding molecules), to form a cyclic template.

[0200] 43. The method according to item 41 or 42, wherein the cyclic amplification template is formed by the ligation of a further oligonucleotide molecule (or two different further oligonucleotide molecules). 44. The method according to any one of items 1 to 43, further comprising the steps of adding two different binding molecules to a biological material and / or adding further binding molecules, followed by blocking nonspecific binding sites of the molecules. 45. The method according to any one of items 1 to 44, wherein the step of detecting nucleic acid ends includes a step of amplifying nucleic acid and producing an amplified product to be detected. 46. ​​The method described in item 45, wherein nucleic acid amplification is rolling circle amplification.

[0201] 47. The process of detecting co-localization between nucleic acid-binding proteins and nucleic acid ends is a. Proximity ligation assay; b. Branched proximity hybridization assay; c. FRET detection; or d. Proximity-driven reactions with phosphors or dyes The method described in any one of items 1 through 46, including the method described in item 1.

[0202] 48. A kit for detecting nucleic acid ends in biological materials, a) Nucleic acid binding molecules; b) A binding molecule that binds to the nucleic acid binding molecule; and c) Binding molecules that bind to nucleic acid-binding proteins A kit that includes this. 49. A kit according to item 48, comprising a proteinase, preferably proteinase K.

[0203] 50. A kit for detecting nucleic acid ends in biological materials, a) Nucleic acid binding molecules; b) A monoclonal antibody that binds to the nucleic acid-binding molecule; and c) Monoclonal antibodies that bind to nucleic acid-binding proteins A kit that includes this.

[0204] 51. A method for evaluating the effectiveness of a therapeutic agent, a) A step of performing the method described in any one of items 1 to 47 on a sample obtained from a subject before administering the therapeutic agent; b) A step of performing the method described in any one of items 1 to 47 on a sample obtained from a subject after administering a therapeutic agent; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where the therapeutic agent is effective if the amount of nucleic acid ends detected in step a) is greater than that detected in step b)). A method comprising, where steps a) and b) can be carried out in any order.

[0205] 52. The method described in item 51, wherein the therapeutic agent targets proteins involved in the mismatch repair (MMR) pathway. 53. The method described in item 51 or 52, wherein the therapeutic agent targets PMS1, PMS2, or MLH1.

[0206] 54. A method for evaluating the effectiveness of a therapeutic agent, a) A step of performing the method described in any one of items 1 to 47 on a sample obtained from a subject before administering the therapeutic agent; b) A step of performing the method described in any one of items 1 to 47 on a sample obtained from a subject after administering a therapeutic agent; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where, if the amount of nucleic acid ends is greater in step b) than in step a), the therapeutic agent is effective). A method comprising, where steps a) and b) can be carried out in any order.

[0207] 55. The method described in item 54, wherein the therapeutic agent targets RPA or RAD51. 56. The method described in item 54 or 55, wherein the sample is a cancer biopsy. 57. The method described in any one of items 54-56, wherein the sample contains cancer cells. 58. The method described in any one of items 54-57, wherein the therapeutic agent is an oncology drug. 59. The method according to any one of items 54-58, wherein the therapeutic agent inhibits DNA synthesis, induces nucleic acid damage, and / or inhibits DNA damage repair.

[0208] 60. A method for evaluating drug-induced nucleic acid damage, a) A step of performing any one of the methods described in item 1 to 47 on a sample obtained from a subject before administration of the drug or exposure to the drug; b) A step of performing the method described in any one of items 1 to 47 on a sample obtained from a subject that has been administered a drug or has been exposed to a drug; c) A step to compare the amount of nucleic acid ends detected in steps a) and b) (where, if the amount of nucleic acid ends is greater in step b) than in step a), the drug causes nucleic acid damage). A method comprising, where steps a) and b) can be carried out in any order.

[0209] 61. A method for detecting single-stranded nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating a biological material with a proteinase to increase the accessibility of the nucleic acid ends to a level obtainable by using a proteinase (e.g., proteinase K) at a concentration of 6 μg / mL to 8 μg / mL for less than 60 seconds; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. A method that may include this.

[0210] 62. The method according to item 61, wherein a nucleic acid-binding protein binds to the end of a single-stranded nucleic acid. 63. The method according to item 61 or 62, wherein the nucleic acid-binding protein is a protein associated with MMR. 64. The method according to any one of items 61-63, wherein the nucleic acid-binding protein is PMS1, PMS2, and / or MLH1.

[0211] 65. A method for detecting double-stranded nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating a biological material with a proteinase to increase the accessibility of the nucleic acid ends to a level obtainable by using a proteinase (e.g., proteinase K) at a concentration of 6 μg / mL to 8 μg / mL for less than 60 seconds; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. A method that may include this.

[0212] 66. The method described in item 65, wherein a nucleic acid-binding protein binds to the ends of a double-stranded nucleic acid. 67. The method according to item 65 or 66, wherein the nucleic acid-binding protein is a protein involved in HR or double-stranded DNA damage repair. 68. The method according to any one of items 65-67, wherein the nucleic acid-binding protein is RAD51 and / or RAD70. [Brief explanation of the drawing]

[0213] These and other embodiments of the present invention will be described with reference to the attached drawings.

[0214] [Figure 1] An exemplary method of the present invention using PMS2 as a nucleic acid-binding protein is shown. [Figure 2] An exemplary method of the present invention is shown, using PMS2 as a nucleic acid-binding protein and further binding molecules. [Figure 3] This invention illustrates an exemplary method in which PMS2 is used as a nucleic acid-binding protein to directly incorporate nucleic acid-binding molecules into a single-stranded DNA strand. [Figure 4] An exemplary method of the present invention using Rad51 as a nucleic acid-binding protein is shown. [Figure 5A] The results for sample 3-NT cells and sSTRIDE MMR (Example 1) staining are shown. [Figure 5B] The results for cells treated with sample 4-6TG and stained with sSTRIDE MMR (Example 1) are shown. [Figure 5C]The results of antigen retrieval 1 (proteinase K, 2 minutes) by 6TG treatment are shown.

[0215] [Figure 6A] The results for sample 5—untreated U2OS WT, stained with sSTRIDE MMR—are shown. [Figure 6B] The results for sample 6—untreated U2OS WT cells and a negative control (anti-biotin antibody only) stained with sSTRIDE MMR—are shown. [Figure 6C] The results for sample 7—untreated U2OS WT cells and a negative control (PMS2 antibody only) stained with sSTRIDE MMR—are shown. [Figure 6D] The results for sample 8—untreated U2OS WT cells—using the sSTRIDE standard assay are shown. [Figure 7] The results of the antigen retrieval test 2 (proteinase K, 1 minute) are shown. [Figure 8] This shows a comparison between the sSTRIDEMMR assay and the negative control. [Figure 9A] The results for sample 9—untreated U2OS WT cells—from the sSTRIDE MMR standard assay are shown. [Figure 9B] The results for sample 10—untreated U2OS WT cells—and the negative control (PMS2 antibody only) of the sSTRIDE MMR assay are shown. [Figure 9C] The results for sample 11—untreated U2OS WT cells—as a positive control (standard procedure) for the sSTRIDE MMR assay are shown. [Figure 9D] The analysis results for antigen retrieval 3 (without proteinase K) are shown. [Figure 10A] This image shows a comparison of three antigen retrieval methods (U2OS WT untreated cells). It is a representative image showing the labeling results performed using the sSTRIDE-MMR assay under different antigen retrieval conditions. [Figure 10B] The results of the analysis of dependence on antigen retrieval are shown.

[0216] [Figure 11] This shows a comparison of focal intensity and frequency. The upper panel shows sSTRIDE MMR foci, and the lower panel shows sSTRIDE assay foci. [Figure 12A] The intensity and frequency of foci (U2OS WT cells, 48-hour treatment, antigen retrieval 2) are shown. [Figure 12B] Comparison of antigen retrieval methods - Abcam antibody is shown. Samples 27-30 - U2OS WT cells treated with different concentrations of 6TG for 48 hours, sSTRIDE MMR positive control (antigen retrieval method v2) [Figure 13A] Comparison of Antigen Retrieval Methods - Results of Quantitative Analysis - This section presents a comparison of antigen retrieval methods. [Figure 13B] The results of the analysis of the ratio between sSTRIDE MMR and sSTRIDE standard operation are shown. [Figure 13C] The results for sample 29-U2OS WT untreated cells - antigen retrieval method v5 are shown. [Figure 14A] Negative control - Abcam antibody shown. Samples 36-39 - Untreated U2OS WT, 48 hours, sSTRIDE MMR assay negative control (antigen retrieval method v2). Labeled reagents were omitted in each enzymatic reaction. [Figure 14B] Quantitative analysis results - Negative control (antigen retrieval v2) are shown. Quantitative analysis results - Comparison of antigen retrieval methods. [Figure 14C] Analysis results - showing the ratio between negative control and sSTRIDE MMR. In the control sample, -N1-PLA, N2-nucleotide (nts), N3-polI reaction, N4-antibody (abs), N5-anti-PMS2, N6-antibiotin [Figure 15A] The results for untreated U2OS WT cells labeled with samples 40-43-sSTRIDE-MMR (40-42) and sSTRIDE (43) are presented, and different antigen retrieval methods were tested. [Figure 15B]Quantitative analysis results - showing the results of antigen retrieval. Analysis results - ratio between sSTRIDE standard procedure and sSTRIDE MMR. [Figure 15C] The results for different antigen retrieval methods are shown.

[0217] [Figure 16A] Negative control - shows the impact of endogenous biotin blockade time on readout levels. Quantitative analysis results - comparison of controls using different biotin blockade times - the red box represents a blockade time of 30 minutes, and the box represents a blockade time of 15 minutes. [Figure 16B] The results for samples 46 and 52-U2OS WT cells and the sSTRIDE MMR assay negative control are shown. Differences at the readout level depend on the blockade time. [Figure 17A] The results of the comparison of antigen retrieval methods are shown below: v2 - 60 seconds, v4 - 15 seconds, v5 - 30 seconds, v6 - 90 seconds. [Figure 17B] The comparison results based on the ratio of sSTRIDE MMR / sSTRIDE are shown. [Figure 18A] The results of comparing the sSTRIDE MMR / sSTRIDE (Abcam antibody) ratio in the negative control are shown. In the control group, the following are omitted: N1-PLA, N2-nucleotide, N3-polI reaction, N4-antibody, N5-anti-PMS2, N6-antibiotin [Figure 18B] The results of comparing the sSTRIDE MMR / sSTRIDE (Santa Cruz Biotechnology antibody) ratio in the negative control are shown. [Figure 19] This shows the evaluation of HAP1 parental cell lines versus PMS2 knockout cell lines using IF staining (Abcam antibody). The representative image shows the overlap of DAP1 and PMS2 signals. [Figure 20] This shows sSTRIDE-MMR labeled HAP1 parent strains versus PMS2 knockout strains.

[0218] [Figure 21]HAP1 parent strain: Untreated positive control and negative technical control (*6TG 1μM treated for 48 hours) are shown. [Figure 22] HAP1 PMS2-KO: Shows the untreated positive control and negative technical control (*6TG 1μM treated for 48 hours). [Figure 23] HAP1 PMS2-KO: Shows untreated positive and negative technical controls (treated differently). [Figure 24] HAP1 parent strain: Shows increased 6TG concentration control. [Figure 25] The images show dSTRIDE with and without etoposide. [Figure 26] The results of dSTRIDE signal data analysis after treatment with etoposide show the level of double-strand DNA break detection. [Figure 27] Analysis of dSTRIDE signal data after etoposide treatment shows the level of double-strand DNA break detection. *Under vehicle conditions, 5% of the majority of damaged cells have 192 or more foci. [Figure 28] The results of dSTRIDE signal data analysis after treatment with etoposide show the level of double-strand DNA break detection. [Figure 29] Analysis of dSTRIDE signal data after etoposide treatment shows the level of double-strand DNA break detection. *Under vehicle conditions, 5% of the majority of damaged cells have 205 or more foci. [Figure 30] The results of dSTRIDE signal data analysis show a comparison of cell lines.

[0219] [Figure 31] This shows RAD51-dSTRIDE staining in HR-deficient and HR-highly expressing cells. [Figure 32] This shows RAD51-dSTRIDE staining in HR-high-expression cell lines with and without etoposide. [Figure 33]dSTRIDE HR signal data analysis and measurement results: Shows the level of double-strand DNA breaks associated with Rad51 after etoposide treatment. [Figure 34] dSTRIDE HR signal data analysis and measurement results: Shows the level of double-strand DNA breaks associated with Rad51 after etoposide treatment. [Figure 35] dSTRIDE HR signal data analysis and measurement results: Shows the level of double-strand DNA breaks associated with Rad51 after etoposide treatment. [Figure 36] dSTRIDE HR signal data analysis and measurement results: Shows the level of double-strand DNA breaks associated with Rad51 after etoposide treatment. [Figure 37] The results of the dSTRIDE HR signaling data analysis show a comparison of cell lines. [Figure 38] This shows dSTRIDE-RPA staining in HR-high-expression cell lines with and without etoposide. [Figure 39] dSTRIDE RPA signal data analysis and measurement results: Shows the level of double-strand DNA breaks associated with RPA70 after etoposide treatment. [Figure 40] dSTRIDE RPA signal data analysis and measurement results: Shows the level of double-strand DNA breaks associated with RPA70 after etoposide treatment.

[0220] [Figure 41] dSTRIDE RPA signal data analysis and measurement results: Shows the level of double-strand DNA breaks associated with RPA70 after etoposide treatment. [Figure 42] dSTRIDE RPA signal data analysis and measurement results: Shows the level of double-strand DNA breaks associated with RPA70 after etoposide treatment. [Figure 43] The results of dSTRIDE RPA signal data analysis show a comparison of cell lines. [Figure 44]This paper compares dSTRIDE, dSTRIDE-HR(Rad51), and dSTRIDE-RPA in etoposide-treated HR-high-expression cell lines. [Figure 45] This paper compares dSTRIDE, dSTRIDE-HR(Rad51), and dSTRIDE-RPA in etoposide-treated HR-deficient cell lines. [Figure 46] The results of quantitative analysis of sSTRIDE-MLH1 in HAP1 cells (WT and KO) are shown. [Figure 47] The results of quantitative analysis of sSTRIDE-MLH1 in HAP1 cells treated with and without 6TG are shown. [Figure 48] The results for PMS2 and MLH1 are shown in comparison. [Figure 49] The results of quantitative analysis of sSTRIDE in HAP1 cells (WT and KO) are shown. [Figure 50] The results for PMS2, PMS1, and MLH1 are shown for comparison.

[0221] Examples The present invention will be described in more detail using the following examples. These examples do not limit the scope of the present invention in any way. The examples will be described based on a general protocol relating to the main embodiment presented in this description.

[0222] Example 1 - Detection protocol for PMS1, PMS2, or MLH1 Sample pretreatment - optional (Optional) Immobilization of biological materials in 70% (v / v) ethanol Proteinase treatment 1. Treatment of biological materials with proteinase K at a concentration of approximately 7 μg / mL for 30 seconds at approximately 22°C (i.e., room temperature) (1:350 dilution of 2.5 mg / mL proteinase K stock concentration in PBS). 2. (Optional) Incubation of 1 mM PMSF in PBS at approximately 22°C for 5 minutes. 3. (Optional) Rinse with PBS DNA terminal modification - optional 1. (Optional) T4 PNK reaction Endogenous biotin blockade process - optional 1. Incubation at approximately 22°C for 30 minutes in (optional) streptavidin-blocking solution. 2. (Optional) Rinse with PBS 3. Incubation at approximately 22°C for 30 minutes in (optional) biotin-blocking solution. 4. (Optional) Rinse with PBS Biotin-modified nucleotide binding 5. Polymerase I reaction at 37°C using biotin-7-ATP, biotin-16-dUTP, biotin-16-dCTP, and dDTP of polymerase I enzyme. 6. Rinse with (optional) buffer antibody binding 1. (Optional) Incubation at 37°C for 60 minutes in Navinci isolation buffer. 2. Incubation of a 1:100 rabbit monoclonal anti-PMS2 (or PMS1 or MLH1) antibody in a Navinci primary antibody dilution at approximately 22°C for 60 minutes. 3. (Optional) Rinse with PBS 4. Incubation of 1:100 mouse monoclonal anti-biotin antibody in a Navinci primary antibody dilution at approximately 22°C for 60 minutes. 5. (Optional) Rinse with PBS PLA operation 1. PLA probe binding reaction at 37°C (Navinci PLA reagent and standard protocol) 2. Reaction A at 37℃ 3. Reaction B at 37℃ 4. Reaction C at 37℃

[0223] List of reagents used in Examples 1-3 [Table 1] Table 1. Reagent Details

[0224] [Table 2] Table 2. Details of the antibody

[0225] Example 2 - Rad51 Detection Protocol Sample pretreatment - optional (Optional) Immobilization of biological materials in 70% (v / v) ethanol Proteinase treatment 1. Treatment of biological materials with proteinase K at a concentration of approximately 7 μg / mL for 60 seconds at approximately 22°C (i.e., room temperature) (1:350 dilution of 2.5 mg / mL proteinase K stock concentration in PBS). 2. (Optional) Incubation of 1 mM PMSF in PBS at approximately 22°C for 5 minutes. 3. (Optional) Rinse with PBS DNA terminal modification - optional 1. (Optional) T4 PNK reaction TdT reaction - Incorporation of BrdU 1. TdT reaction at 37°C using BrdU and TdT enzymes 2. (Optional) Rinse with PBS antibody binding 6. (Optional) Incubation at 37°C for 60 minutes in Navinci blockade buffer. 7. Incubation of 1:100 rabbit monoclonal anti-Rad51 antibody in a Navinci primary antibody dilution at approximately 22°C for 60 minutes. 8. (Optional) Rinse with PBS 9. Incubation of 1:100 mouse monoclonal anti-BrdU antibody in a Navinci primary antibody dilution at approximately 22°C for 60 minutes. 10. (Optional) Rinse with PBS PLA operation 1. PLA probe binding reaction at 37°C (Navinci PLA reagent and standard protocol) 2. Reaction A at 37℃ 3. Reaction B at 37℃ 4. Reaction C at 37℃

[0226] Example 3 - RPA detection protein Sample pretreatment - optional 1. Immobilization of (optional) biological materials in 70% (v / v) ethanol. Proteinase treatment 1. Treatment of biological materials with proteinase K at a concentration of approximately 7 μg / mL for 60 seconds at approximately 22°C (i.e., room temperature) (1:350 dilution of 2.5 mg / mL proteinase K stock concentration in PBS). 2. (Optional) Incubation of 1 mM PMSF in PBS at approximately 22°C for 5 minutes. 3. (Optional) Rinse with PBS DNA terminal modification - optional 1. (Optional) T4 PNK reaction TdT reaction - Incorporation of BrdU 1. TdT reaction at 37°C using BrdU and TdT enzymes 2. (Optional) Rinse with PBS antibody binding 1. Incubation at 37°C for 60 minutes in (optional) Navinci blockade buffer. 2. Incubation of 1:100 rabbit monoclonal anti-RPA70 antibody in a Navinci primary antibody dilution at approximately 22°C for 60 minutes. 3. (Optional) Rinse with PBS 4. Incubation of 1:100 mouse monoclonal anti-BrdU antibody in a Navinci primary antibody dilution at approximately 22°C for 60 minutes. 5. (Optional) Rinse with PBS PLA operation 1. PLA probe binding reaction at 37°C (Navinci PLA reagent and standard protocol) 2. Reaction A at 37℃ 3. Reaction B at 37℃ 4. Reaction C at 37℃

[0227] Example 4 - Optimization of protease treatment Objective of the experiment Experimental setup and optimization: Detection and quantitative analysis of PMS2-activated DNA single-strand breaks in human U2OS WT cells method U2OS cells U2OS cells were cultured in DMEM HG medium (Gibco) containing 10% FBS (Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin, using T25 Falson (Nunc) culture medium. Antigen retrieval method - Proteinase K treatment Proteinase K was used after opening a new vial. The antigen retrieval method numbers correspond to the numbers shown in the table below. [Table 3]

[0228] PMS2 antibody Rabbit recombinant anti-PMS2 antibody [EPR3947] - BSA and azide-free (ab214442) Mouse monoclonal anti-PMS2 antibody (B-3): sc-25315

[0229] Test conditions - 5 optimization experiments [Table 4]

[0230] The sSTRIDE method followed all the steps of Example 1, except that it did not rely on the detection of endogenous nucleic acid binding molecules and instead focused solely on the detection of nucleic acid ends. Thus, in the sSTRIDE method, the standard procedure is to apply proteinase K treatment (the same concentration and temperature as in Example 1) for 3 minutes. The (optional) step of endogenous biotin blockade is performed for 15 minutes each, instead of 30 minutes each. Two anti-biotin antibodies are used instead of the anti-PMS2 antibody and anti-biotin antibody (each with a different affinity for biotin molecules and / or a different binding site). The same reagents were used in each experiment (see the reagents in Example 1). The streptavidin / biotin endogenous biotin kit was the same in each experiment. The same batch of proteinase K was used for each sample.

[0231] Staining procedure Abcam antibody The sample was processed using the sSTRIDE (above) standard protocol, modified as follows: 1. Different antigen retrieval methods were tested. 2. One of the biotin antibodies was replaced with a PMS2 antibody (assay sSTRIDE MMR). 3. A positive control was performed (sSTRIDE standard assay was conducted). 4. A negative control was established (one antibody was discarded, and incubation proceeded with a 1% BSA solution without the antibody).

[0232] [Table 5] Table 3. Details of the method; Rb-rabbit antibody (host), Ms-mouse antibody (host)

[0233] Staining procedure: Santa Cruz Biotechnology antibody The samples were processed using the sSTRIDE standard protocol, modified as follows: 1. Different antigen retrieval methods were tested. 2. One of the biotin antibodies was replaced with a PMS2 antibody (assay sSTRIDE MMR). 3. A positive control was performed (sSTRIDE standard assay was conducted). 4. A negative control was established (one antibody was discarded, and incubation proceeded with a 1% BSA solution without the antibody).

[0234] [Table 6] Table 4. Details of the method; Rb-rabbit antibody (host), Ms-mouse antibody (host)

[0235] As a result, Part 1 [Table 7] Table 5. Test conditions

[0236] Figure 5A - Sample 3 - NT cells, stained with sSTRIDE MMR (Example 1); a small number of foci were observed. The maximum number of foci was 3 per nucleus (antigen retrieval 1). Figure 5B - Cells treated with sample 4-6TG, stained with sSTRIDE MMR (Example 1); a small number of foci were observed. The maximum number of foci was 3 per nucleus (antigen retrieval 1). Figure 5C - Analysis Results - Antigen Retrieval 1 (Proteinase K, 2 minutes); Quantitative image analysis shows a small number of detectable foci and no response observed after 6TG treatment.

[0237] [Table 8] Table 6. Test conditions

[0238] Figure 6A - Sample 5 - Untreated U2OS WT, sSTRIDE MMR staining; more foci are observed with antigen retrieval method 2 compared to antigen retrieval method 1. A small amount of background signal is detected (antigen retrieval 2). Figure 6B - Sample 6 - Untreated U2OS WT cells, sSTRIDE MMR stained negative control (anti-biotin antibody only); single focus may be detected (antigen retrieval 2). Figure 6C - Sample 7 - Untreated U2OS WT cells, sSTRIDE MMR stained negative control (PMS2 antibody only); no focus detected, high background signal observed (antigen retrieval 2). Figure 6D - Sample 8 - Untreated U2OS WT cells, sSTRIDE standard assay; multiple foci in a single nucleus - more than 20 foci may be detected per nucleus (antigen retrieval 2).

[0239] Figure 7 - Analysis Results - Antigen Retrieval 2 (Proteinase K, 1 min); Quantitative image analysis shows a comparison of readouts in U2OS cells labeled with sSTRIDE and STRIDE-MMR assays with readouts in the negative control. The results show that sSTRIDE-MMR foci constitute approximately 12% of the total SSB, while the signal in the negative control remains at a low level. Figure 8 - Comparison between the sSTRIDE MMR assay and the negative control; samples were stained and imaged on the same day. Focus intensity and frequency are compared (U2OS WT cells, NT, antigen retrieval 2). The image shows that very few foci were detected in the negative control when one of the antibodies (targeting biotin or PMS2) was absent.

[0240] [Table 9] Table 7. Test conditions

[0241] Figure 9A - Sample 9 - Untreated U2OS WT cells, sSTRIDE MMR standard assay; a single focus may be detected in the nucleus, and high background intensity is observed (antigen retrieval 3). Figure 9B - Sample 10 - Untreated U2OS WT cells, sSTRIDE MMR assay negative control (PMS2 antibody only); no signal detected (antigen retrieval 3). Figure 9C - Sample 11 - Untreated U2OS WT cells, sSTRIDE MMR assay positive control (standard procedure); more than 10 signals per nucleus may be detected (antigen retrieval 3). Figure 9D - Analysis Results - Antigen Retrieval 3 (without proteinase K): The results of quantitative image analysis show that when proteinase treatment is absent, the number of foci detected by sSTRIDE-MMR (PMS2) and sSTRIDE is small.

[0242] Summary - Part 1 In the study, Method 2 (incubating with proteinase K for 1 minute) was found to be the best antigen retrieval method for sSTRIDE MMR PMS2. Focuses were barely detectable with Methods 1 and 3.

[0243] Figure 10A - Comparison of three antigen retrieval methods (U2OS WT untreated cells); This is a representative image showing the labeling results performed by the sSTRIDE-MMR assay using different antigen retrieval conditions. Different numbers of fluorescence foci can be observed under various antigen retrieval conditions, with the largest number of foci being visualized under antigen retrieval v2. Figure 10B - Analysis Results - Dependence on Antigen Retrieval; Quantitative image analysis confirmed that the number of foci was highest when Method 2 was applied. The sSTRIDE assay without proteinase (antigen retrieval 3) is less effective than the assay using proteinase conditions. The number of foci per nucleus decreases by 90% after 1 minute of proteinase treatment (antigen retrieval 3). Background levels from the PMS2 antibody are negligible in each assay condition. Background levels from biotin antibodies are not negligible in the assay. The average number of foci per nucleus is 0.9, but in some nuclei, the number is greater than 5. Differences in the number and morphology of foci were detected through the assay.

[0244] Figure 11 - Comparison of focal intensity and frequency; the upper panel shows sSTRIDE MMR foci, and the lower panel shows sSTRIDE assay foci. sSTRIDE MMR foci are highly intense but infrequent (U2OS WT cells, NT, antigen retrieval 1).

[0245] Conclusion, Part 1 1. The antigen retrieval method has a significant impact in the sSTRIDE standard assay and the sSTRIDE MMR2 assay. 2. The antigen retrieval step is relevant in the sSTRIDE MMR PMS2 assay: assays without proteinase do not function. 3. The best test method for antigen retrieval is to use proteinase K for 1 minute. 4. Background levels from biotin antibodies are not negligible - a negative control using biotin antibodies is relevant for each sSTRIDE MMR PMS2 staining.

[0246] As a result, Part 2 [Table 10] [Table 11] Table 8. Test conditions

[0247] Samples 15-18 and 21 show a maximum of 2-3 foci per nucleus, therefore 3D is not inferred, and for this reason, these conditions cannot be used as a positive control for the assay.

[0248] Conclusion, Part 2 6TG-treated cells did not respond as expected. Readout levels were lower than those in untreated samples. Antigen retrieval method 2 was used under each condition. However, in some cells, the foci were very bright and large, which leads to the conclusion that MMR has high intensity at specific spots and multiple foci are detected.

[0249] Figure 12A - Focus intensity and frequency - (U2OS WT cells, 48-hour treatment, antigen retrieval method 2); the morphology of the foci is as expected, with a high proportion of signal to noise. Figure 12B - Comparison of antigen retrieval methods - Abcam antibody; Samples 27-30 - U2OS WT cells treated with different concentrations of 6TG for 48 hours, sSTRIDE MMR assay positive control (antigen retrieval method v2). Treatment did not significantly increase the number of nuclear foci.

[0250] Figure 13A - Comparison of antigen retrieval methods; Results of quantitative analysis - Comparison of antigen retrieval methods Figure 13B - Analysis results - Percentage between sSTRIDE MMR and sSTRIDE standard operation; the most promising antigen retrieval method is v5-30 seconds. In this sample, the signal distribution was different, with some groups of nuclei having more than 40 foci per nucleus. Figure 13C - Sample 29 - U2OS WT untreated cells - Antigen retrieval method v5; Antigen retrieval method v5 was selected as the best antigen retrieval method.

[0251] Figure 14A - Negative control - Abcam antibody; Samples 36-39 - U2OS WT untreated cells, 48 ​​hours, sSTRIDE MMR assay negative control (antigen retrieval method v2). Reagent labeling was omitted in each enzymatic reaction. Figure 14B - Results of quantitative analysis - Negative control (antigen retrieval 2); Results of quantitative analysis - Comparison of antigen retrieval methods.

[0252] The graph does not show the results derived from sample 37. The number of SSBs per nucleus is 3.8. The number of foci in the approximate control is negligible. However, the number of SSBs in the negative control using polymerase I suggests that the endogenous biotin blockade time should be extended in this assay. The ratio between the sSTRIDE MMR assay and the control also shows a significant signal in the negative control where polymerase I enzyme was omitted (the ratio is 0.49, although this is not shown in the plot). Figure 17C - Analysis results - Percentage between negative control and sSTRIDE MMR; in the control sample, N1-PLA, N2-nucleotide (nts), N3-polI reaction, N4-antibody (abs), N5-anti-PMS2, and N6-anti-biotin are omitted.

[0253] As a result, Part 3 [Table 12] [Table 13] Table 9. Test conditions

[0254] In sample 39, the maximum number of foci observed per nucleus was 2-3, so 3D was not inferred, and therefore this condition could not be used as a positive control for the assay. Samples 52 and 53 were stained with Abcam antibody. In this procedure, the endogenous biotin blockade time was changed to 30 minutes (15 minutes longer than in the previous experiment).

[0255] Figure 15A - Samples 40-43 - Untreated U2OS WT cells labeled with sSTRIDE-MMR(40-42) and sSTRIDE(43) were tested using different antigen retrieval methods. Figure 15B - Results of quantitative analysis - Antigen retrieval; Analysis results - Ratio between sSTRIDE standard procedure and sSTRIDE MMR Figure 15C - Experiment 05 tested the most promising version of the antigen retrieval procedure. Antigen retrieval v5 showed the highest signal per nucleus (the same version was selected in the experiment using Abcam antibody).

[0256] Figure 16A - Negative control - Effect of endogenous biotin blockade time on readout levels; Results of quantitative analysis - Comparison of controls using different biotin blockade times - Red boxes indicate a blockade time of 30 minutes, and no box indicates a blockade time of 15 minutes. Figure 16B - Samples 46 and 52 - U2OS WT cells, sSTRIDE MMR assay negative control; differences between readout levels depending on blockade time.

[0257] Figure 17A - Comparison - Antigen Retrieval Methods; v2 - 60 seconds, v4 - 15 seconds, v5 - 30 seconds, v6 - 90 seconds: The highest readout was observed with 30 seconds of treatment using proteinase K. Higher readout was observed with Santa Cruz Biotechnology antibodies. Figure 17B-sSTRIDE: Comparison of MMR / sSTRIDE ratios; the ratio is highest for both antibodies after 30 seconds of treatment with antigen retrieval v5-proteinase K.

[0258] Figure 18A-sSTRIDE MMR / sSTRIDE (Abcam antibody) negative control ratio comparison; in the control group, N1-PLA, N2-nucleotide, N3-polI reaction, N4-antibody, N5-anti-PMS2, and N6-antibiotin are omitted. The ratios were at very low levels in all negative controls, confirming the specificity of the assay. Figure 18 shows a comparison of the negative control ratios for B-sSTRIDE MMR / sSTRIDE (Santa Cruz Biotechnology antibody). This ratio is higher than that for Abcam antibody, indicating that Santa Cruz Biotechnology antibody is characterized by more nonspecific binding.

[0259] In conclusion, Part 3 Both Abcam and Santa Cruz Biotechnology antibodies can be used in the sSTRIDE-MMR assay. The highest readout was obtained with Santa Cruz Biotechnology, which also yielded a higher readout in the negative control. Increasing the endogenous biotin blockade time may help reduce the background noise observed in the negative control.

[0260] Example 5 - Development of a variant of the sSTRIDE-MMR(PMS2) assay A. Cell culture conditions HAP1 strain The HAP1 cell line is a human-like haploid cell line derived from chronic myeloid leukemia (CML). Advantages of the HAP1 cell line in assay development studies include: being a single copy of any specific allele, having a rapid doubling time, and being easy to transfect. The HAP1 parental and HAP1 PMS2 knockout cell lines are provided as isogeneic pairs. The PMS2 protein knockout was achieved by editing the PMS2 coding exon to include a 1 bp insertion via CRISPR / Cas. HAP1 cells and HAP1 PMS2 KO cells were purchased from the Horizon Discovery cell culture collection (May 18, 2022). A detailed description of the cell culture is listed in Table 10. Cells were cultured in T75 flasks containing IMDM + 10% FBS + 1% penicillin-streptomycin solution at 37°C and 5% CO2 (10,000 units each).

[0261] The master bank collection was prepared from #1 passage cultures performed at the intoDNA Laboratory. The banking transactions were carried out as shown below. The cell suspension was centrifuged at room temperature (1000 rpm, 5 minutes). Next, the medium was removed and a sterile mixture of 60% medium, 30% FBS, and 10% DMSO was added. The mixture was gently mixed and dispensed into cryotubes using a pipette (800k cells are stored in one bank). The cryotubes were transferred to Nalgene Mr. Frosty freezing containers and slowly frozen at -80°C (temperature was lowered by approximately 1°C per minute).

[0262] The experiments included in this report were conducted using a cell bank prepared on June 6, 2022. [Table 14] Table 10. List of cell banks purchased from ATCC

[0263] B. Sowing and Processing Protocols Sowing protocol When cell confluence reaches 90%, the cells are sterilized with PBS (Ca 2+ and Mg 2+ The cells were washed (without EDTA) and incubated with 0.25% trypsin solution (2 mL) containing EDTA for 2 minutes. The trypsin was inactivated with FBS-containing medium, the cells were collected, and counted in a Barker chamber. The cells were then seeded on coverslips of 12-well plates at the densities (including passage number) listed in Table 11. The cells were then left to stand in an incubator for 24 hours.

[0264] [Table 15] Table 11. List of cell seeding densities. The same density was used for sSTRIDE and sSTRIDE MMR samples. Passages are those performed at the intoDNA Institute.

[0265] Processing protocol After standing for 24 hours, the working medium was replaced with fresh medium supplemented with 1 μM or 10 μM 6-thioguanine (6TG) in DMSO and incubated for 48 hours (the final DMSO concentration was 0.1% in the medium under each experimental condition). Under vehicle conditions, the working medium was replaced with fresh medium supplemented with 0.1% sterile DMSO and incubated for the incubation period. Next, all samples were fixed with 70% EtOH cooled on ice and stored at -20°C.

[0266] *The 6TG stock was newly prepared from powder. The powder was suspended in sterile DMSO to a final stock concentration of 100 mM. The 6TG in DMSO was stored in the dark at -20°C.

[0267] C. Reagents [Table 16] Table 12. List of reagents used in cell culture procedures.

[0268] D. Antibody List [Table 17] Table 13. List of antibodies used in assay validation.

[0269] In assays using MLH1 and PMS1, antigen retrieval was tested for 30 seconds. E. Cell culture conditions U2OS cells U2OS cells were purchased from the ATCC cell culture collection via intoDNA. The cells were cultured in T25 flasks containing DMEM HG + 10% FBS + 1% penicillin-streptomycin solution (10,000 units each) at 37°C with 5% CO2.

[0270] The banking transaction was performed as follows: The cell suspension was centrifuged at room temperature (1000 rpm, 5 minutes). Next, the medium was removed and a sterile mixture (60% medium, 30% FBS, and 10% DMSO) was added. The mixture was gently mixed and dispensed into cryotubes using a pipette (800k cells were stored in one bank). The cryotubes were transferred to Nalgene Mr. Frosty freezing containers and slowly frozen at -80°C (temperature was lowered by approximately 1°C per minute).

[0271] F. Sowing and Processing Protocols Sowing protocol When cell confluence reaches 90%, the cells are sterilized with PBS (Ca 2+ and Mg 2+The cells were washed (without EDTA) and incubated with 1 mL of 0.25% trypsin solution containing EDTA for 4 minutes. The trypsin was inactivated with FBS-containing medium, the cells were collected, and counted in a Barker chamber. The cells were then seeded on coverslips of 12-well plates at the densities (including passage number) listed in Table 14. The cells were then left to stand in an incubator for 24 hours.

[0272] [Table 18] Table 14. List of cell seeding densities. The same density was used for sSTRIDE and sSTRIDE MMR samples. Passage numbers are those performed at the intoDNA Institute.

[0273] Processing protocol After standing for 24 hours, the working medium was replaced with fresh medium supplemented with 6-thioguanine (6TG)** in DMSO and allowed to stand for 24 or 48 hours of incubation (the final DMSO concentration was 0.1% in the medium under each experimental condition). Under vehicle conditions, the working medium was replaced with fresh medium supplemented with 0.1% sterile DMSO and allowed to stand for the incubation period. Next, all samples were fixed with ice-cold 70% EtOH and stored at -20°C.

[0274] * The 6TG concentration ranged from 250 nM to 50 μM. The diluent (DMSO) concentration in the culture medium of each well was 0.1%. **The 6TG stock was newly prepared from powder. The powder was suspended in sterile DMSO to a final stock concentration of 100 mM. The 6TG in DMSO was stored in the dark at -20°C.**

[0275] G. Reagents [Table 19] Table 15. List of reagents used in cell culture procedures.

[0276] H. sSTRIDE-MMR (PMS2) labeling Labeling was performed according to the protocol described in Example 1.

[0277] I. Results Figure 19 shows the evaluation of HAP1 parental cell lines versus PMS2 knockout cell lines using IF staining (Abcam antibody). Representative images show the overlap of DAP1 and PMS2 signals. Loss or very weak signaling from the PMS2 channel confirms that one of the cell lines is in a knockout state. Figure 20 shows sSTRIDE-MMR-labeled HAP1 parental strain vs. PMS2 KO cells. Fluorescence foci are visible in HAP1 WT cells, but not in most HAP1 PMS2 KO cells. Figure 21 shows the HAP1 parent strain: untreated positive control and negative technical control (*6TG 1 μM treated for 48 hours). Quantitative image analysis shows that 6TG treatment results in an increase in the number of detected fluorescence foci, while the negative control sample shows a very small number of detected foci.

[0278] Figure 22 shows the HAP1 PMS2-KO:untreated positive control and negative technical control (*6TG 1 μM 48-hour treatment). Quantitative image analysis shows that 6TG treatment does not result in a significant increase in the number of fluorescence foci in HAP1 PMS2 KO cells, while a very small number of foci are detected in the negative control sample. Figure 23 shows HAP1 PMS2-KO cells: untreated positive control and negative technical control. The histogram of frequency distribution highlights the differences in sSTRIDE-MMR readouts between cell lines. Figure 24 shows the increase in 6TG concentration control in the HAP1 parent strain. Quantitative image analysis indicates that treatment with higher concentrations of 6TG results in higher levels of sSTRIDE-MMR signaling.

[0279] conclusion 1) Comparing sSTRIDE-MMR(PMS2) readouts between HAP1 WT and PMS2 KO cells reveals a significant difference in the number of foci detected in these cell lines. As expected, fewer foci were detected in the HAP1 PMS2 KO cell line. 2) The specificity of this assay is confirmed by the small number of foci detected in HAP1 PMS2 KO cells. 3) Treatment with 6TG resulted in a statistically significant increase in the number of sSTRIDE-MMR foci only in HAP1 WT cells.

[0280] Example 6 - Comparison of dSTRIDE, dSTRIDE-RAD51, and dSTRIDE-RPA readouts in NCI-H661 (i.e., HR high expression) and NCI-H1693 (i.e., HR deficient) cell lines. Cell culture conditions NCI-H1693 and NCI-H661 were purchased from the ATCC cell culture collection (May 18, 2022). A detailed description of the cell culture is listed in Table 16. Cells were cultured in T75 flasks containing RPMI1640 + 10% FBS + 1% penicillin-streptomycin solution (10,000 units each) at 5% CO2 and 37°C.

[0281] The Master Bank Collection was prepared from the first generation. The banking operations were performed as follows: The cell suspension was centrifuged at room temperature (1000 rpm, 5 minutes). Next, the culture medium was removed, and a sterile mixture of 60% medium, 30% FBS, and 10% DMSO was added. The mixture was gently mixed and dispensed into cryotubes using a pipette (800k cells were stored in one bank). The cryotubes were transferred to a Nalgene Mr. Frosty freezer and slowly frozen at -80°C (temperature was lowered by approximately 1°C per minute). After 120 minutes, the cryotubes were transferred to liquid nitrogen.

[0282] The experiments included in this report were conducted using a cell bank prepared on May 30, 2022. [Table 20] Table 16. List of cell banks purchased from ATCC

[0283] Seeding and processing protocols Sowing protocol When cell confluence reaches 90%, the cells are sterilized with PBS (Ca 2+ and Mg 2+ The cells were washed (without EDTA) and incubated with 0.25% trypsin solution (2 mL) containing EDTA for 2 minutes. The trypsin was inactivated with FBS-containing medium, the cells were collected, and counted in a Barker chamber. The cells were then seeded on coverslips of 12-well plates at the densities (including passage number) listed in Table 17. The cells were then left to stand in an incubator for 24 hours.

[0284] [Table 21] Table 17. List of cell seeding densities: The same density was used for dSTRIDE, dSTRIDE HR, and dSTRIDE RPA under each treatment condition.

[0285] Processing protocol After standing for 24 hours, the working medium was replaced with fresh medium supplemented with 5 μM or 50 μM etoposide* and incubated for 48 hours (the final DMSO concentration was 0.1% in the medium under each experimental condition). Under vehicle conditions, the working medium was replaced with fresh medium supplemented with 0.1% sterile DMSO and incubated for the incubation period. Next, all samples were fixed with ice-cold 70% EtOH and stored at -20°C. *Etoposide stock (100 mM in DMSO) should be stored in a dark place at -20°C.

[0286] reagent [Table 22] Table 18. List of reagents used in cell culture procedures.

[0287] dSTRIDE / dSTRIDE-RAD51 / dSTRIDE-RPA labeling Labeling was performed according to the protocol described in Example 2 or 3.

[0288] result 1) In both cell lines, etoposide treatment resulted in a dose-dependent increase in the number of double-strand DNA breaks detected by dSTRIDE. 2) The baseline level of DSBs (dSTRIDE) and the increase in DSBs after 5 μM etoposide treatment were similar in both cell lines. 3) 50 μM etoposide resulted in a more significant increase in the number of double-stroke bonds (dSTRIDE) in the NCI-H1693 (i.e., HR-deficient) cell line. 4) In both cell lines, etoposide treatment resulted in a dose-dependent increase in the number of RPA-related double-strand DNA breaks detected by dSTRIDE-RPA. 5) Treatment with 50 μM etoposide resulted in a significant increase in the number of RAD51-related double-strand DNA breaks detected by dSTRIDE-RAD51 in NCI-H661 (i.e., a cell line with high HR expression capacity). 6) Etoposide treatment did not result in a detectable increase in the number of RAD51-related double-strand DNA breaks detected by dSTRIDE-RAD51 in NCI-H1693 (i.e., HR-deficient cell line).

[0289] Figure 25 shows dSTRIDE images with and without etoposide. Representative images show an increase in the number of double-strand DNA breaks detected by dSTRIDE after etoposide treatment and represented as fluorescence foci in its HR-deficient cell line (i.e., NCI-H1693). Figure 26 shows the level of double-strand DNA breaks measured after dSTRIDE signal data analysis following etoposide treatment. Quantitative image analysis shows that the number of DSBs increases in a dose-dependent manner after etoposide treatment.

[0290] Figure 27 shows the level of double-strand DNA breaks measured by dSTRIDE signal data analysis after etoposide treatment. *Under vehicle conditions, 5% of the majority of damaged cells have 192 or more foci. Figure 28 shows the level of double-strand DNA breaks measured after dSTRIDE signal data analysis following etoposide treatment. Quantitative image analysis shows that the number of DSBs increases in a dose-dependent manner after etoposide treatment. Figure 29 shows the level of double-strand DNA breaks measured by dSTRIDE signal data analysis after etoposide treatment. *Under vehicle conditions, 5% of the majority of damaged cells have 205 or more foci. Figure 30 shows a comparison of cell lines based on dSTRIDE signal data analysis. Quantitative image analysis shows that more double-stroke syndromes (DSBs) are induced in HR-deficient cell lines than in HR-high-expression cell lines after treatment with 50 μM etoposide.

[0291] Figure 31 shows RAD51IF staining in HR-high expression and HR-deficient cell lines. No clear differences were observed between the cell lines. Figure 32 shows -dSTRIDE-HR(RAD51) staining in HR-high-expression cell lines with and without etoposide. Etoposide treatment results in an increase in the number of visible fluorescence foci. Figures 33-36 show the levels of double-strand DNA breaks associated with Rad51, as determined by the analysis of dSTRIDE HR signaling data after etoposide treatment. High-HR cell lines show a dose-dependent increase in the number of dSTRIDE-HR(RAD51) detected after etoposide treatment, while HR-deficient cell lines show no response.

[0292] Figure 37 shows the results of dSTRIDE HR signal data analysis and cell line comparison. No response was observed in HR-deficient cell lines. Figure 38 shows dSTRIDE-RPA staining of HR-high-expression cell lines with and without etoposide. An increase in the number of foci is observed after treatment with etoposide. Figures 39-42 show the results of dSTRIDE RPA signal data analysis after etoposide treatment, indicating the measurement level of double-strand DNA breaks associated with RPA70. The results show a dose-dependent increase in the number of dSTRIDE-RPA foci after etoposide treatment in both cell lines. Figure 43 shows a comparison of cell lines based on the results of dSTRIDE RPA signal data analysis. The results indicate that more dSTRIDE-RPA foci are detected in HR-deficient cell lines after treatment with 50 μM etoposide.

[0293] Figure 44 shows a comparison of dSTRIDE, dSTRIDE-HR(Rad51), and dSTRIDE-RPA in HR-high-expressing cell lines treated with etoposide. The results show a dose-dependent response in all assays after etoposide treatment. Figure 45 shows a comparison of dSTRIDE, dSTRIDE-HR(Rad51), and dSTRIDE-RPA in HR-deficient cell lines treated with etoposide. Quantitative image analysis revealed that etoposide treatment led to dose-dependent formation of double-strand DNA breaks in HR-deficient cells. Results from the dSTRIDE-RPA assay provided evidence that RPA is involved in the repair of these lesions, while results from the dSTRIDE-HR(RAD51) assay confirmed that the recruitment of RAD51 to the DSB site was inefficient.

[0294] conclusion Etoposide efficiently induces DSBs in both cell lines tested, but mutations in the HR pathway in the HR-deficient (i.e., NCI-H1693) cell line result in increased cleavage levels. The inability of this cell line to perform normal HR repair is evident from the following: when dSTRIDE, dSTRIDE-RPA, and dSTRIDE-RAD51 data are combined, the latter assay shows no response, confirming insufficient RAD51 loading. Thus, the dSTRIDE-RAD51 assay can be considered a functional HR assay that provides information about the state of HR repair. This information can be used in a method to predict whether tumors from subjects diagnosed with cancer have the ability to repair DNA by homologous recombination. Specifically, tumor samples taken from subjects can be analyzed for RAD51 and / or RAD-70 protein levels using the STRIDE protocol described herein. If the levels are lower than expected compared to control cell samples, the tumor sample may contain a deficiency in proteins involved in HR repair. If the amount is equal to or higher than expected compared to a control cell sample, the tumor sample may not contain a deficiency in the protein involved in HR repair. This information can also be used to select an appropriate anticancer agent (e.g., a PARP inhibitor) to treat the subject. In addition, this information can be used to predict the response of a subject diagnosed with cancer to anticancer treatment, particularly when the anticancer treatment targets proteins involved in the HR pathway (or mismatch repair pathway). This information can also be used to select a tailored treatment for a subject diagnosed with cancer, particularly when the treatment includes a molecule that targets proteins involved in the HR pathway (or mismatch repair pathway). For example, that molecule may be a PARP inhibitor.

[0295] Example 7 - Development of the sSTRIDE-MMR (PMS1 and MLH1) assay The sSTRIDE-MMR method was carried out in the same manner as in Examples 1 and 5. However, instead of targeting PMS2, either PMS1 or MLH1 was targeted.

[0296] Figure 46 shows the results of quantitative analysis of sSTRIDE-MLH1 in HAP1 cells (WT and KO). Quantitative image analysis shows that the MLH1 protein is present in the SSB of both PMS2 WT and KO cell lines, and that 6TG treatment results in an increase in sSTRIDE-MMR(MLH1) readout. Figure 47 shows the results of quantitative analysis of sSTRIDE-MLH1 in HAP1 cells treated with and without 6TG. The frequency distribution plot confirms the readout from the sSTRIDE-MMR(MLH1) assay. Figure 48 shows a comparison of results between PMS2 and MLH1. The results indicate that PMS2 is present at very low levels in HAP1 PMS2 KO cells, while MLH1 can be recruited to SSB in both cell lines.

[0297] Figure 49 shows the results of quantitative analysis of sSTRIDE-PMS1 in HAP1 cells (WT and KO). The results indicate that PMS1 is present in the SSB in both cell lines and participates in the MMR process after treatment with 6TG. Figure 50 shows a comparison of the results for PMS2, PMS1, and MLH1. The results indicate that both MLH1 and PMS2 are involved in MMR in the absence of PMS2.

[0298] Example 8 - dSTRIDE-HR (RAD51 and RPA) assay for cancer biopsy samples The dSTRIDE-HR method was performed in the same manner as in Examples 2 and 3 on breast cancer biopsy samples from patients who were (a) free of BRCA mutations (i.e., wild-type), (b) had mutations in the BRCA1 gene, and (c) had mutations in the BRCA2 gene. The samples were independently tested for RAD51 (according to the protocol of Example 2) and RPA (according to the protocol of Example 3). The results show that levels of RAD51- and RPA-binding DNA ends are significantly lower in samples from patients with BRCA1 and BRCA2 gene mutations compared to levels measured in samples from patients without BRCA mutations (wild-type). This indicates a deficiency in the homologous recombination pathway in samples with BRCA1 and BRCA2 gene mutations. Samples lacking the HR pathway are considered suitable for treatment with anticancer drugs, such as PARP inhibitors.

Claims

1. A method for detecting nucleic acid ends in a biological material containing nucleic acids, a) A step of incubating the biological material with proteinase for less than two minutes; b) A step of adding a nucleic acid binding molecule to a biological material under conditions such that the nucleic acid binding molecule binds to the nucleic acid ends within the biological material; c) The process of attaching two different binding molecules to a biological material under conditions such that one binding molecule binds to a nucleic acid binding molecule attached to the nucleic acid terminal, and the other binding molecule binds to a nucleic acid binding protein that binds to the nucleic acid at the nucleic acid terminal; and d) A process for detecting nucleic acid ends in biological material by detecting the co-localization of nucleic acid-binding proteins and nucleic acid ends via the binding of two different binding molecules. Methods that include...

2. The method according to claim 1, wherein the nucleic acid comprises DNA and / or RNA, preferably DNA.

3. The method according to claim 1 or claim 2, wherein the nucleic acid end is a single-stranded nucleic acid cleavage or a double-stranded nucleic acid cleavage.

4. The method according to any one of claims 1 to 3, wherein the proteinase is aspartate protease, glutamate protease, metalloprotease, cysteine ​​protease, serine protease, or threonine protease, and preferably the protease is a serine protease such as proteinase K.

5. Incubation of biological materials with proteinases: a. Between 1 second and less than 2 minutes, between 15 seconds and 90 seconds, between 20 seconds and 80 seconds, between 25 seconds and 70 seconds, preferably between about 30 seconds and about 60 seconds; and / or b. At a temperature between 15°C and 30°C, preferably between 20°C and 25°C. The method according to any one of claims 1 to 4, which is implemented.

6. The method according to any one of claims 1 to 5, wherein the proteinase is present in the solution at a concentration preferably of 1 μg / mL to 20 μg / mL, 3 μg / mL to 15 μg / mL, 5 μg / mL to 10 μg / mL, or 6 μg / mL to 8 μg / mL, preferably at a concentration of about 7 μg / mL.

7. nucleic acid binding molecules: a. Halogenated nucleotide or nucleoside molecule, DNA precursor analog, and / or biotinylated nucleotide molecule; and / or b. For example, binding to nucleic acid ends within biological materials by an addition process using an enzyme catalyst employing DNA polymerase I, TdT, Klenow fragment, Phu polymerase, Taq polymerase, T4 DNA polymerase, T7 DNA polymerase, T4 polynucleotide kinase, or RNA polymerase. The method according to any one of claims 1 to 6.

8. a. The two different binding molecules may be, or contain, two different monoclonal antibodies or their fragments, and / or b. Two different binding molecules each attach to different oligonucleotide molecules. The method according to any one of claims 1 to 7.

9. Nucleic acid-binding proteins: a. Proteins found in biological materials; b. Proteins that bind to nucleic acids directly or indirectly at the nucleic acid terminus; c. Nucleic acid repair proteins, preferably nucleic acid end (e.g., cleavage) repair proteins; and / or d. RAD51, RPA (e.g., RPA70), PMS2, MLH1, PMS1, p53, MSH2, ataxia telangiectasia and Rad3-related proteins, ATM serine / threonine kinase, RAD52, XRCC1, proliferating cell nuclear antigen, XPC, Ku70, Ku80, nibrin, DDB2, Bloom syndrome protein, CHEK2, RAD51C, DNA polymerase eta, Rad50, D DB1, RBBP8, FANCB, PALB2, H2AX, yH2AX DNA repair and recombinant protein RAD54-like, Primpol, REV1, terminal deoxynucleotidyl transferase, DNA polymerase NY, Fanconi anemia, complementation group C, FANCF, ERCC8, Artemis, ubiquitin ligase, RNF4, TP53BP1, AP endonuclease, ERCC4, transcription factor II H, XRCC3, XRCC2, RecA, ERCC6, SLX4, sirtuin 1, PTEN, replication protein A2, replication protein A3, Alkb homolog 3, alpha-ketoglutarate-dependent dioxygenase, exonuclease 5, DNA polymerase alpha catalytic subunit, cyclin H, PARP1 / 2. The method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 9, further comprising the step of adding two different binding molecules, each of which binds to the two different binding molecules, wherein the two different further binding molecules may optionally each attach to a different oligonucleotide molecule.

11. The method according to any one of claims 1 to 10, further comprising the step of adding a different further oligonucleotide molecule to the two different binding molecules (or to the further binding molecule) after the step of adding two different binding molecules (or, if applicable to claim 10, after the step of adding a further binding molecule), to form a cyclic template, wherein the cyclic template is optionally formed by the ligation of the two different further oligonucleotide molecules.

12. The method according to any one of claims 1 to 11, wherein the detection of nucleic acid ends includes a step of amplifying nucleic acid to produce a detected amplification product, wherein the nucleic acid amplification is optionally rolling circle amplification.

13. Detection of co-localization between nucleic acid-binding proteins and nucleic acid terminals, a. Proximity ligation assay; b. Branched proximity hybridization assay; c. FRET detection; or d. Proximity-driven reactions with phosphors or dyes A method according to any one of claims 1 to 12, including the method described in any one of claims 1 to 12.

14. A kit for detecting nucleic acid ends in biological materials, a) Nucleic acid binding molecules; b) A binding molecule that binds to the nucleic acid binding molecule; and c) Binding molecules that bind to nucleic acid-binding proteins A kit that includes this.

15. A method for evaluating the effectiveness of a therapeutic agent, a) The step of performing the method according to any one of claims 1 to 13 on a sample obtained from a subject before administering the therapeutic agent; b) The step of performing the method according to any one of claims 1 to 13 on a sample obtained from a subject after administering the therapeutic agent; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where the therapeutic agent is effective if the amount of nucleic acid ends detected in step a) is greater than that detected in step b)). Includes, Here, steps a) and b) can be carried out in any order. The therapeutic agent here may optionally target PMS1, PMS2, or MLH1; or a) The step of performing the method according to any one of claims 1 to 13 on a sample obtained from a subject before administering the therapeutic agent; b) The step of performing the method according to any one of claims 1 to 13 on a sample obtained from a subject after administering the therapeutic agent; c) A step to compare the amounts of nucleic acid ends detected in steps a) and b) (where the therapeutic agent is effective if the amount of nucleic acid ends is greater in step b) than in step a). Includes, Here, steps a) and b) can be carried out in any order. The therapeutic agent here is optionally targeted at RPA or RAD51. method.

16. The use of a kit for detecting nucleic acid ends in biological materials, wherein the kit a) Nucleic acid binding molecules; b) A binding molecule that binds to the nucleic acid binding molecule; and c) Binding molecules that bind to nucleic acid-binding proteins Including its use.

17. The kit according to claim 14 or the use according to claim 16, wherein the kit further comprises a proteinase.

18. The kit or use according to claim 17, wherein the proteinase is proteinase K.

19. A method for predicting the response of a subject diagnosed with cancer to anti-cancer treatment, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by carrying out the method according to any one of claims 1 to 13; and (ii) predicting the response of the subject to anti-cancer treatment based on the level of nucleic acid-binding proteins measured in the sample.

20. A method for selecting a customized therapy for a subject diagnosed with cancer, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by carrying out the method according to any one of claims 1 to 13; and (ii) selecting a customized therapy for the subject based on the level of nucleic acid-binding proteins measured in the sample.

21. A method for predicting whether a tumor derived from a subject diagnosed with cancer is capable of DNA repair by homologous recombination, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from the subject by carrying out the method according to any one of claims 1 to 13; and (ii) predicting, based on the level of nucleic acid-binding proteins measured in the sample, whether a tumor derived from a subject diagnosed with cancer is capable of DNA repair by homologous recombination.

22. A method for evaluating the state of mismatch repair pathways in a sample containing tumor cells obtained from a subject, comprising: (i) measuring the level of nucleic acid-binding proteins in tumor cells by carrying out the method according to any one of claims 1 to 13; and (ii) evaluating the state of mismatch repair pathways based on the level of nucleic acid-binding proteins measured in tumor cells.

23. A method for classifying subjects diagnosed with cancer into a patient cohort, comprising: (i) measuring the level of nucleic acid-binding proteins in a sample obtained from a subject by carrying out the method according to any one of claims 1 to 13; and (ii) classifying the subjects diagnosed with cancer into a patient cohort based on the level of nucleic acid-binding proteins measured in the sample.

24. The method according to any one of claims 19 to 23, wherein the nucleic acid-binding protein is RAD51, RPA70, PMS1, PMS2, or MLH1.

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