Method for detecting nucleic acid end(s)

Description

METHOD FOR DETECTING THE END(S) OF NUCLEIC ACID FIELD

[001] The invention relates to methods for detecting nucleic acid end(s) in a biological material containing nucleic acids. The invention also relates to kits for detecting nucleic acid end(s) in a biological material and associated uses of the kits. In addition, the invention relates to methods for evaluating the efficacy of a therapeutic agent. FUNDAMENTALS

[002] Since the early days of the concept of synthetic lethality in response to DNA damage, the homologous recombination (HR) state of cancer cells has been the focus of attention for researchers and clinicians. Homologous repair deficiency (HRD) can be defined by a low RAD51 score. For example, a low RAD51 score has emerged as an indicator of patient prognosis and response to therapeutic agents used in the treatment of triple-negative breast cancer. A low RAD51 score is also used to assess the therapeutic efficacy of inhibitors of PARP. Although there are different approaches to detect RAD51 (e.g., RAD51 immunofluorescence (IF)), functional biomarkers capable of assessing RAD51-mediated HR proficiency are lacking. Petition 870250109009, dated 11 / 27 / 2025, page 10 / 249 2 / 190

[003] Mismatch repair (MMR) is considered one of the fundamental pathways that protect genome stability, and mutations in genes encoding crucial MMR proteins (e.g., PMS1, PMS2, or MLH1) have been shown to promote cancer initiation. Conversely, inactivation of MMR components in cancer cells leads to an increase in neoantigen formation and, consequently, exposes the tumor to the immune system. Therapeutic strategies aimed at inhibiting MMR are therefore under development; however, methods that report the MMR status are lacking.

[004] Document WO2019 / 035727 A1 describes a method for detecting DNA end(s). The method is based on molecules, such as BrdU, that bind directly to the DNA end(s), followed by the binding of binding molecules (e.g., antibodies) to the molecules bound to the DNA end(s). In the method, the binding molecules are conjugated to oligonucleotides capable of hybridizing with additional oligonucleotides to form a circular structure. The circular structure provides a template for rolling circle amplification. Once amplified, the amplification product can be detected by known detection methods in the art, e.g., fluorescence microscopy. However, this method does not allow the direct detection of nucleic acid-binding proteins that Petition 870250109009, dated 11 / 27 / 2025, p. 11 / 249 3 / 190 are involved in the response to nucleic acid damage (e.g., DNA), in mismatch repair, and in homologous recombination.

[005] Thus, there is a need for more precise approaches to detect proteins involved in the response and repair of nucleic acid damage (e.g., DNA), mismatch repair, and homologous recombination. SUMMARY OF THE INVENTION

[006] The inventors have modified the method in document WO2019 / 035727 A1 (called “STRIDE”) to allow 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 end(s) (e.g., DNA ends) in biological material. The inventors have encountered a precise method for detecting and quantifying proteins involved in nucleic acid (e.g., DNA) damage response and repair, mismatch repair, and homologous recombination. Thus, the approach provides a method for the simultaneous detection of the nucleic acid end(s) and the nucleic acid-binding protein(s) bound to the nucleic acid at that end(s). Notably, this method includes the detection of cytosolic nucleic acids (especially DNA), which may be Petition 870250109009, dated 11 / 27 / 2025, p. 12 / 249 4 / 190 linked, for example, to cyclic GMP-AMP synthase (cGAS).

[007] In one aspect, the invention provides a method for detecting nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different binding molecules to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to the nucleic acid-binding protein that is attached to the nucleic acid at or near the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[008] Incubation of biological material with a proteinase improves the detection of a binding protein to Petition 870250109009, dated 11 / 27 / 2025, p. 13 / 249 5 / 190 nucleic acid in biological material (compared to the situation where no proteinase is used). Specifically, incubating biological material with a proteinase increases the accessibility of the nucleic acid end(s) without significantly affecting the levels of nucleic acid-binding protein in the biological material.

[009] Without being tied to any theory, increasing the accessibility of the nucleic acid end(s) may minimize steric hindrance in locating the DNA ends to be detected.

[0010] It should be understood that each aspect and embodiment that refers to a method for detecting nucleic acid end(s) in biological material also refers to a method for detecting nucleic acid-binding proteins that are bound to the nucleic acid at or near the nucleic acid end(s). Thus, each aspect and embodiment described herein can be understood as referring to a method for detecting a nucleic acid-binding protein in a biological material comprising the nucleic acid-binding protein.

[0011] The step of incubating the biological material with a proteinase can be performed under such conditions to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a Petition 870250109009, dated 11 / 27 / 2025, p. 14 / 249 6 / 190 concentration of about 7 pg / mL (e.g., 7.1 pg / mL) for less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); Incubation with proteinase can be carried out with reference to any of examples 1 to 3.

[0012] The invention provides a method for detecting nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL for less than 2 minutes at a temperature of approximately 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different binding molecules to the biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the end(s) of the nucleic acid and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the Petition 870250109009, dated 11 / 27 / 2025, p. 15 / 249 7 / 190 nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0013] The invention provides a method for detecting nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of about 7 pg / mL (e.g., 7.1 pg / mL) for less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different linker molecules to biological material under conditions such that one linker molecule binds to nucleic acid linker molecules attached to the nucleic acid end(s) and the other linker molecule binds to a linker protein. Petition 870250109009, dated 11 / 27 / 2025, p. 16 / 249 8 / 190 a nucleic acid that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0014] The invention provides a method for detecting single-stranded nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid ends to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 60 seconds at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different linker molecules to the biological material under conditions such that one of the linker molecules binds to the nucleic acid linker molecules attached to the nucleic acid end(s) and the Petition 870250109009, dated 11 / 27 / 2025, p. 17 / 249 9 / 190 another binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the end(s) of the nucleic acid; and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules, where the nucleic acid-binding protein binds to the single-stranded nucleic acid end(s).

[0015] The invention provides a method for detecting double-stranded nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) add two different bonding molecules Petition 870250109009, dated 11 / 27 / 2025, p. 18 / 249 10 / 190 to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules, where the nucleic acid-binding protein binds to the end(s) of the double-stranded nucleic acid.

[0016] The invention provides a method for detecting nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase for less than 2 minutes; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different binding molecules to biological material under conditions such that one of them Petition 870250109009, dated 11 / 27 / 2025, p. 19 / 249 11 / 190 binding molecules bind to nucleic acid-binding molecules attached to the nucleic acid end(s), and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0017] The invention provides a method for detecting single-stranded nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase for less than 60 seconds; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different linker molecules to biological material under conditions such that one linker molecule binds to nucleic acid linker molecules attached to the nucleic acid end(s) and the other linker molecule binds to a linker protein. Petition 870250109009, dated 11 / 27 / 2025, p. 20 / 249 12 / 190 a nucleic acid that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules, where the nucleic acid-binding protein binds to the single-stranded nucleic acid end(s).

[0018] The invention provides a method for detecting double-stranded nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase for less than 2 minutes; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different binding molecules to the biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the end(s) of the nucleic acid and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the Petition 870250109009, dated 11 / 27 / 2025, p. 21 / 249 13 / 190 nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules, where the nucleic acid-binding protein binds to the end(s) of the double-stranded nucleic acid.

[0019] In all aspects and modalities described in this document, nucleic acids preferably comprise or consist of DNA.

[0020] In all aspects and modalities described in this document, preferably, the nucleic acid end(s) are the DNA end(s).

[0021] In all aspects and modalities described in this document, the proteinase is preferably proteinase K.

[0022] In all aspects and embodiments described in this document, preferably, the nucleic acid-binding protein and the nucleic acid-binding molecule are not the same molecules. Thus, the nucleic acid-binding protein and the nucleic acid-binding molecule are preferably different molecules.

[0023] In one aspect, the invention provides a kit for detecting nucleic acid end(s) in a material Petition 870250109009, dated 11 / 27 / 2025, p. 22 / 249 14 / 190 organic, the kit includes: a) nucleic acid-binding molecules; b) a linker molecule that binds to nucleic acid-binding molecules; and c) a binding molecule that binds to a nucleic acid-binding protein.

[0024] In one aspect, the invention provides a kit for detecting nucleic acid end(s) in a biological material, the kit comprising: a) nucleic acid-binding molecules; b) a monoclonal antibody that binds to nucleic acid-binding molecules; and c) a monoclonal antibody that binds to a nucleic acid-binding protein.

[0025] Preferably, the kit also includes a proteinase (e.g., proteinase K).

[0026] In one aspect, the invention provides a method for evaluating the effectiveness of a therapeutic agent, the method comprising the steps of: a) perform the detection method described in this document on a sample obtained from an individual before administration of the therapeutic agent, b) perform the detection method described in this document on a sample obtained from the individual after administration of the therapeutic agent, Petition 870250109009, dated 11 / 27 / 2025, page 23 / 249 15 / 190 c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the therapeutic agent is effective if the amount of nucleic acid end(s) detected is greater in step a) than in step b), where steps a) and b) can be performed in any order.

[0027] Preferably, the therapeutic agent targets PMS1, PMS2, or MLH1.

[0028] In one aspect, the invention provides a method for evaluating the effectiveness of a therapeutic agent, the method comprising the steps of: a) perform the detection method described in this document on a sample obtained from an individual before administration of the therapeutic agent, b) perform the detection method described in this document on a sample obtained from the individual after administration of the therapeutic agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the therapeutic agent is effective if the amount of nucleic acid end(s) detected is greater in step b) than in step a), where steps a) and b) can be performed in any order. Petition 870250109009, dated 11 / 27 / 2025, page 24 / 249 16 / 190

[0029] Preferably, the therapeutic agent targets RPA (e.g., RPA70) or RAD51.

[0030] In one aspect, the invention provides a method for evaluating nucleic acid damage caused by an agent, the method comprising the steps of: a) perform the detection method described in this document on a sample obtained from an individual prior to administration of, or exposure to, an agent, b) perform the detection method described in this document on a sample obtained from the individual after administration of, or exposure to, an agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the agent causes damage to the nucleic acid if the amount of nucleic acid end(s) detected is greater in step b) than in step a), where steps a) and b) can be performed in any order.

[0031] In one aspect, the invention provides a method for predicting the response of an individual diagnosed with cancer to an anticancer treatment, comprising (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual by performing the detection method as described in this document and (ii) predicting the individual's response to treatment. Petition 870250109009, dated 11 / 27 / 2025, page 25 / 249 17 / 190 anticancer based on the determined level of nucleic acid-binding protein in the sample.

[0032] In one aspect, the invention provides a method for selecting a personalized therapy for an individual diagnosed with cancer, comprising (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual by performing the detection method as described in this document; and (ii) selecting a personalized therapy for the individual based on the determined level of the nucleic acid-binding protein in the sample.

[0033] In one aspect, the invention provides a method for classifying an individual diagnosed with cancer into a cohort of patients, comprising (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual by performing the detection method as described in this document; and (ii) classifying an individual diagnosed with cancer into a cohort of patients based on the determined level of the nucleic acid-binding protein in the sample.

[0034] In one aspect, the invention provides a method for predicting whether a tumor in an individual diagnosed with cancer is capable of repairing DNA by homologous recombination, comprising (i) determining the level of a nucleic acid-binding protein in a sample obtained Petition 870250109009, dated 11 / 27 / 2025, page 26 / 249 18 / 190 of the individual performing the detection method as described in this document; and (ii) predict whether a tumor in an individual diagnosed with cancer is capable of repairing DNA by homologous recombination based on the determined level of nucleic acid-binding protein in the sample.

[0035] In one aspect, the invention provides a method for evaluating the state of a mismatch repair pathway in a sample comprising a tumor cell obtained from an individual, the method comprising (i) determining the level of a nucleic acid-binding protein in the tumor cell by performing the detection method as described in this document; and (ii) evaluating the state of the mismatch repair pathway based on the determined level of the nucleic acid-binding protein in the tumor cell.

[0036] In one aspect, the invention provides a medicament for use in the treatment of cancer in an individual, wherein the individual has been identified as responsive to said medicament by one of the methods described in this document.

[0037] Each aspect or modality as defined in this document may be combined with any other aspect(s) or embodiment(s), unless clearly indicated otherwise. In particular, any feature indicated as being preferred or advantageous Petition 870250109009, dated 11 / 27 / 2025, p. 27 / 249 19 / 190 can be combined with any other feature or features indicated as being preferred or advantageous.

[0038] The inventors have surprisingly discovered that biological material (e.g., a cell) comprising a nucleic acid and a nucleic acid-binding protein can be subjected to proteinase treatment to increase the accessibility of the nucleic acid end(s) without significantly affecting the levels of the nucleic acid-binding protein in the biological material. The increased accessibility of the nucleic acid end(s) facilitates the binding of nucleic acid-binding molecules to the nucleic acid end(s), which improves the detection level of the nucleic acid end(s) and nucleic acid-binding proteins in the biological material. Thus, the inventors have come up with a more accurate and sensitive method for detecting nucleic acid-binding proteins (and colocalized nucleic acid end(s)) in biological material. DETAILED DESCRIPTION

[0039] Unless otherwise defined in this document, the scientific and technical terms used in connection with the present invention shall have the meanings normally understood by a person skilled in the art. Petition 870250109009, dated 11 / 27 / 2025, page 28 / 249 20 / 190 The meaning and scope of the terms should be clear; however, in the case of any latent ambiguity, the definitions provided in this document prevail over any dictionary definition or extrinsic definition.

[0040] It should be understood that singular articles, such as a, an, and the, are frequently used for convenience; however, all singular occurrences are intended to encompass the plural unless otherwise indicated, either explicitly or by context. The terms comprise, include, and have are inclusive and mean that there may be additional elements beyond those listed. Furthermore, it should be understood that all references, including journal articles, books, patents, technical documents, and the like mentioned in this description, are incorporated herein by reference in their entirety and for all purposes.

[0041] The term about, as used in this document for numerical data, refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and the use of the term about at the beginning of a sequence of values ​​modifies each of the values ​​(i.e., about 1, 2 and 3 refers to about 1, about 2 and about 3). For example, a temperature of about 25 °C may include temperatures between (and including) 22.5 °C and 27.5 °C. Petition 870250109009, dated 11 / 27 / 2025, p. 29 / 249 21 / 190

[0042] The invention provides a method for detecting nucleic acid end(s) (e.g., DNA end(s)) in a biological material containing nucleic acids (e.g., DNA). The invention also provides a method for detecting nucleic acid-binding protein(s) in a biological material containing nucleic acid-binding protein(s). The method is based on the presence of a nucleic acid-binding protein i) in the vicinity of; or ii) bound to one or more nucleic acid end(s). The method is based on detecting the colocalization event of the nucleic acid-binding protein and the nucleic acid end(s) in the biological material. Preferably, the nucleic acid-binding protein is found in the biological material. That is, the method is based on the presence of intrinsic nucleic acid-binding proteins in the biological material.

[0043] The requirement that “the nucleic acid-binding protein is bound to the nucleic acid at the nucleic acid end(s)” means that the nucleic acid-binding protein is bound directly or indirectly, in sufficient proximity to the nucleic acid end(s) to permit detection of the colocalization of the nucleic acid-binding protein and the nucleic acid end(s) by the methods described in this document. Sufficient proximity is Petition 870250109009, dated 11 / 27 / 2025, p. 30 / 249 22 / 190 typically within 100 nm of the nucleic acid end(s), preferably within 50 nm of the nucleic acid end(s), and most preferably within 40 nm of the nucleic acid end(s). The nucleic acid-binding protein may be bound to the nucleic acid at the nucleic acid end(s) indirectly, for example, as part of a (multiprotein) complex, which provides sufficient proximity to the nucleic acid end(s) to permit detection. The nucleic acid-binding protein may be bound to cytosolic nucleic acid fragments, for example, in the case of cGAS. As would be readily understood by a person skilled in the art, the term “at the nucleic acid end(s),” as used in this document, is intended to encompass nucleic acid residues that are at the end of a nucleic acid strand. This encompasses gaps within a nucleic acid strand.Preferably, the nucleic acid end(s) are the result of nucleic acid damage, homologous recombination, or mismatch repair.

[0044] The term “intrinsic,” in the context of nucleic acid-binding proteins, is intended to encompass nucleic acid-binding proteins that are found in biological material. Thus, these proteins are not added to the biological material as part of the methods or Petition 870250109009, dated 11 / 27 / 2025, p. 31 / 249 23 / 190 kits described in this document. Intrinsic nucleic acid-binding proteins are naturally found (i.e., naturally present) in biological material. Thus, the biological material (e.g., cell) may comprise a nucleic acid and a nucleic acid-binding protein prior to the proteinase treatment step. Intrinsic nucleic acid-binding proteins are not added to the biological material by a user of the method or kit.

[0045] Colocalization-based detection, as used in this document (in the context of the colocalization of the nucleic acid end(s) and nucleic acid-binding protein), means that the presence of the nucleic acid end(s) and nucleic acid-binding protein is detected based on their proximity. This typically occurs through a single detection signal (e.g., fluorescence) generated only when the nucleic acid end(s) and 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 the nucleic acid end(s). In the absence of this close proximity, no signal is generated. The nucleic acid-binding proteins detected according to the invention can therefore be those involved in the detection or repair of Petition 870250109009, dated 11 / 27 / 2025, page 32 / 249 24 / 190 nucleic acid damage (i.e., damage that results in detectable nucleic acid end(s), which includes cytosolic chromatin fragments).

[0046] Nucleic acid-binding proteins can bind to nucleic acid at the nucleic acid end(s) 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., protein) that binds directly to the nucleic acid and therefore acts as an adapter molecule for the indirect binding of a nucleic acid-binding protein).

[0047] By testing a variety of experimental parameters, the inventors found ideal conditions for detecting the colocalization of nucleic acid-binding proteins and the nucleic acid end(s) (to which the nucleic acid-binding protein binds, directly or indirectly, as defined herein). Specifically, the inventors found that treatment with proteinase (before the detection step) increases the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of nucleic acid-binding protein to be detected. Preferably, the proteinase is a serine proteinase (especially proteinase K). The inventors found that treatment with proteinase at a concentration between 6 Petition 870250109009, dated 11 / 27 / 2025, p. 33 / 249 25 / 190 pg / mL and 8 pg / mL (e.g., about 7 pg / mL) for less than 90 minutes (preferably 90 seconds or less) at a temperature of about 22 °C (i.e., room temperature) provides an ideal combination of increased accessibility of the nucleic acid end(s) and no significant effect on the levels of nucleic acid-binding protein to be detected. A person skilled in the art would understand that these parameters can be altered, for example, by reducing the proteinase concentration and extending the treatment time, or by increasing the proteinase concentration and reducing the treatment time. Proteinase treatment is preferably shorter when the aim is to detect a single-stranded nucleic acid end in the biological material, compared to detecting the double-stranded nucleic acid end(s).The methods described in this document may comprise a step of incubating the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase (e.g., proteinase K) at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 2 minutes (preferably 90 seconds or less) (and without significantly affecting the levels of nucleic acid-binding protein to be detected). If the nucleic acid end(s)... Petition 870250109009, dated 11 / 27 / 2025, p. 34 / 249 26 / 190 nucleic acid for single-stranded nucleic acids, the methods described in this document may comprise a step of incubating the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase (e.g., proteinase K) at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 90 seconds (preferably about 30 seconds) (and without significantly affecting the levels of nucleic acid-binding protein to be detected). Preferably, the method is carried out under conditions such that the levels of nucleic acid-binding proteins to be detected are not significantly reduced.

[0048] Expressions not significantly affected, not significantly reduced, or not significantly reduced, as used in this document in the context of nucleic acid-binding protein levels, mean that the detectable levels of the nucleic acid-binding protein(s) after proteinase treatment remain substantially the same as before treatment. For example, the detectable levels of the nucleic acid-binding protein(s) 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%, Petition 870250109009, dated 11 / 27 / 2025, p. 35 / 249 27 / 190 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 pre-treatment levels. Preferably, nucleic acid-binding protein levels remain detectable and, due to the reduction of background proteins, are detected more efficiently than in the absence of proteinase treatment.

[0049] Biological material may comprise a nucleic acid (e.g., DNA). Biological material may comprise a nucleic acid-binding protein (e.g., DNA-binding protein).

[0050] The invention provides a method for detecting nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) add two different bonding molecules Petition 870250109009, dated 11 / 27 / 2025, p. 36 / 249 28 / 190 to biological material under conditions such that one of the binding molecules binds to nucleic acid binding molecules attached to the end(s) of the nucleic acid and the other binding molecule binds to the nucleic acid binding protein that is attached to the nucleic acid at the end(s) of the nucleic acid; and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0051] The invention also provides a method for detecting nucleic acid-binding protein(s) in a biological material containing nucleic acid-binding protein, wherein the method comprises the steps of: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) add two different bonding molecules Petition 870250109009, dated 11 / 27 / 2025, p. 37 / 249 29 / 190 to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to the nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect nucleic acid-binding protein(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0052] As discussed in this document, nucleic acid-binding protein can bind to nucleic acid directly or indirectly.

[0053] Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid binding molecules to biological material under conditions such that the nucleic acid binding molecules bind to the end(s) of Petition 870250109009, dated 11 / 27 / 2025, p. 38 / 249 30 / 190 nucleic acid in biological material; (c) adding two different binding molecules to the biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to the nucleic acid-binding protein that is directly attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules. [0 054] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; Petition 870250109009, dated 11 / 27 / 2025, p. 39 / 249 31 / 190 (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to the nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s) via an adapter molecule (e.g., an adapter protein); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules. [0 055] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase (e.g., proteinase K) for less than 2 minutes; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) add two different bonding molecules Petition 870250109009, dated 11 / 27 / 2025, p. 40 / 249 32 / 190 to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0056] The invention provides a method for detecting single-stranded nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase for less than 60 seconds; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different linker molecules to the biological material under conditions such that one of the linker molecules binds to the nucleic acid linker molecules attached to the nucleic acid end(s) and the Petition 870250109009, dated 11 / 27 / 2025, p. 41 / 249 33 / 190 another binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the end(s) of the nucleic acid; and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules, where the nucleic acid-binding protein binds to the single-stranded nucleic acid end(s).

[0057] The invention provides a method for detecting double-stranded nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase for less than 2 minutes; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different linker molecules to biological material under conditions such that one linker molecule binds to nucleic acid linker molecules attached to the nucleic acid end(s) and the other linker molecule binds to a linker protein. Petition 870250109009, dated 11 / 27 / 2025, p. 42 / 249 34 / 190 a nucleic acid that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules, where the nucleic acid-binding protein binds to the end(s) of the double-stranded nucleic acid.

[0058] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different linker molecules to biological material under conditions such that one of the linker molecules binds to the acid-linking molecules. Petition 870250109009, dated 11 / 27 / 2025, p. 43 / 249 35 / 190 nucleic acid-linked to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0059] The invention provides a method for detecting single-stranded nucleic acid end(s) in a biological material containing nucleic acids, which may comprise the steps of: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid ends to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 60 seconds at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different binding molecules to biological material under conditions such that one of them Petition 870250109009, dated 11 / 27 / 2025, p. 44 / 249 36 / 190 binding molecules bind to nucleic acid-binding molecules attached to the nucleic acid end(s), and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules, where the nucleic acid-binding protein binds to the single-stranded nucleic acid end(s).

[0060] The invention provides a method for detecting double-stranded nucleic acid end(s) in a biological material containing nucleic acids, which may comprise the steps of: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid binding molecules to biological material under conditions such that the nucleic acid binding molecules bind to the end(s) of Petition 870250109009, dated 11 / 27 / 2025, p. 45 / 249 37 / 190 nucleic acid in biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules, where the nucleic acid-binding protein binds to the end(s) of the double-stranded nucleic acid.

[0061] Incubation of biological material with a proteinase can 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.

[0062] Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the acid ends. Petition 870250109009, dated 11 / 27 / 2025, p. 46 / 249 38 / 190 nucleic acid levels achieved 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) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0063] Incubation of biological material with a proteinase can be carried out for 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, between 30 seconds and 60 seconds, preferably incubating the Petition 870250109009, dated 11 / 27 / 2025, p. 47 / 249 39 / 190 biological material with a proteinase is performed for between 30 and 60 seconds.

[0064] The methods, kits, and uses described in this document may focus on the detection of one or more nucleic acid-binding proteins that specifically bind to one or more single-stranded nucleic acid ends (i.e., single-stranded nucleic acid breaks, for example, caused by DNA damage). If nucleic acid-binding proteins that bind to single-stranded nucleic acid breaks (e.g., PMS2) are to be detected, incubation may be performed for between 1 second and less than 90 seconds, between 5 seconds and 75 seconds, between 10 seconds and 60 seconds, or between 15 seconds and 45 seconds. Preferably, incubation is performed for about 30 seconds.

[0065] The methods, kits, and uses described in this document may focus on the detection of one or more nucleic acid-binding proteins that specifically bind to one or more ends of double-stranded nucleic acid (i.e., double-stranded nucleic acid breaks, for example, caused by DNA damage). If nucleic acid-binding proteins that bind to double-stranded nucleic acid breaks (e.g., RAD51 or RPA) are to be detected, incubation may be performed for between 1 second and less than 2 minutes, between 10 seconds and 90 Petition 870250109009, dated 11 / 27 / 2025, pp. 48 / 249 40 / 190 seconds, between 20 seconds and 80 seconds, or between 30 seconds and 70 seconds. Preferably, incubation is carried out for about 60 seconds.

[0066] Shorter proteinase incubation for nucleic acid-binding proteins that bind specifically to one or more single-stranded nucleic acid ends (e.g., PMS1, PMS2, or MLH1), compared to double-stranded nucleic acid ends, is preferred to compensate for the smaller number of these proteins at the nucleic acid end site (e.g., nucleic acid damage). Proteinase incubation time is largely related to the nature of the specific nucleic acid-binding protein; for example, in the case of double-strand breaks, multiple RPA and RAD51 molecules are recruited to the nucleic acid end(s). This number is lower in the case of PMS2 proteins, which are recruited to single-strand breaks.

[0067] Incubation of biological material with a proteinase can be carried out for at least 1 second, at least 5 seconds, preferably at least 10 seconds, and most preferably at least 15 seconds.

[0068] Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: Petition 870250109009, dated 11 / 27 / 2025, pp. 49 / 249 41 / 190 a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for between 1 second and less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0069] Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: Petition 870250109009, dated 11 / 27 / 2025, p. 50 / 249 42 / 190 a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for between 15 and 90 seconds at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0070] Incubation of biological material with a proteinase can be carried out for approximately 30 seconds or approximately 60 seconds. For the detection of the end(s) Petition 870250109009, dated 11 / 27 / 2025, p. 51 / 249 For the detection of single-stranded nucleic acid 43 / 190, the incubation step of the biological material with a proteinase is preferably 90 seconds or less, 60 seconds or less, and even more preferably about 30 seconds. For the detection of double-stranded nucleic acid end(s), the incubation step of the biological material with a proteinase is preferably less than 2 minutes, 90 seconds or less, and even more preferably about 60 seconds.

[0071] Incubation of biological material with a proteinase can be carried out at a temperature between 10 °C and 55 °C, between 15 °C and 55 °C, between 20 °C and 55 °C, between 10 °C and 35 °C or between 15 °C and 30 °C, preferably between 20 °C and 25 °C. Even more preferably, incubation of biological material with a proteinase (e.g., proteinase K) is carried out at a temperature of about 22 °C (i.e., room temperature).

[0072] The proteinase may be in solution. Preferably, the proteinase is at a concentration between 0.1 pg / mL and 100 pg / mL, between 1 pg / mL and 20 pg / mL, between 3 pg / mL and 15 pg / mL, between 5 pg / mL and 10 pg / mL, or between 6 pg / mL and 8 pg / mL, preferably between 6 pg / mL and 8 pg / mL. Preferably, the concentration is less than 10 pg / mL. The proteinase may be at a concentration of about 7 pg / mL. The solution may comprise a buffer, such as PBS. The solution may comprise SDS (e.g., 0.2 to 1% SDS) and / or Petition 870250109009, dated 11 / 27 / 2025, p. 52 / 249 44 / 190 urea (e.g., 1 to 4 M urea). The solution may have a pH between 4.0 and 12.5, preferably between pH 7.0 and 8.0. For example, if the solution comprises PBS, the pH is approximately 7.4.

[0073] The inventors found that incubating biological material with proteinase K as described in this document (i.e., for less than 2 minutes, preferably at a concentration of 6-8 pg / mL) provides more accurate detection of the nucleic acid end(s) than when using proteinase K for 2.5 minutes at a concentration of 10 pg / mL.

[0074] A proteinase can be a broad-spectrum proteinase. The term “broad-spectrum proteinase” is intended to encompass proteinases capable of digesting a wide variety of native proteins. A particularly preferred example of a broad-spectrum proteinase is proteinase K. The main cleavage site of proteinase K is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha-amino groups. Any other proteinase with an identical or similar mode of action would be considered a broad-spectrum proteinase for the purposes of this invention.

[0075] A proteinase can be an aspartyl protease, a glutamyl protease, a metalloprotease, a cysteine ​​protease, a serine protease, or a threonine protease. From Petition 870250109009, dated 11 / 27 / 2025, page 53 / 249 45 / 190 a preferred mode, the protease is a serine protease, such as proteinase K.

[0076] For all aspects and modalities described in this document, the proteinase is preferably proteinase K.

[0077] Incubation of biological material with a proteinase can be interrupted by the addition of a proteinase inhibitor. Any suitable proteinase inhibitor can be used. For example, the proteinase inhibitor can be phenylmethylsulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), or calbiochem. The proteinase inhibitor can be incubated with the proteinase (which is 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 of PMSF can be used for 5 minutes to interrupt the action of the proteinase. After incubation, the proteinase inhibitor can be removed by a washing step (e.g., with a buffer such as PBS). The incubation of biological material with a proteinase can be interrupted by at least one rinsing step.

[0078] The invention provides a method for detecting nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises Petition 870250109009, dated 11 / 27 / 2025, p. 54 / 249 46 / 190 steps of: a) Incubate the biological material with a proteinase for less than 2 minutes; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[0079] Biological material may be, or may be derived from, an animal, a plant, a protozoan, a bacterium, or a virus. Biological material may comprise a cell or tissue, or a fragment thereof. Biological material may comprise a living cell or a fixed cell. The method may comprise a fixation step. Petition 870250109009, dated 11 / 27 / 2025, page 55 / 249 47 / 190 before the incubation step of the biological material with a proteinase. The fixation step may comprise incubation of the biological material with ethanol (e.g., 70% (v / v) ethanol). The fixation step may be carried out for between 1 h and 24 h, between 2 h and 12 h, or between 3 h and 6 h. Preferably, the fixation step is carried out for at least 2 h. The setting stage can be carried out for at least 8 hours. The fixation step can be performed at a temperature between -80 °C and 30 °C or between -40 °C and 25 °C, preferably around -20 °C. The method may further comprise a step of fixing the biological material to the solid support before the step of incubating the biological material with a proteinase. The step of fixing the biological material to the solid support may be performed before or after the fixation step (if present). Preferably, the solid support is a hydrophilic solid support (i.e., a positively and / or negatively charged solid support). A hydrophilic solid support is understood to be a solid support comprising at least one hydrophilic outer surface. Thus, a hydrophilic surface is presented to the biological material, resulting in the fixation of one or more individual cell(s) to the solid support (by means of the hydrophilic surface). The hydrophilic solid support may be a slide or a coverslip.The hydrophilic nature of the solid support can result from a... Petition 870250109009, dated 11 / 27 / 2025, p. 56 / 249 48 / 190 coating on the solid support or of the solid support composition itself. The coating may provide a positively or negatively charged surface, but a positive charge is preferred. For example, the solid support may be a TOMO® slide or a slide coated with poly-L-lysine, both providing a positively charged surface to which cells bind. In some embodiments, the solid support may be an uncoated silicate glass slide. The biological material (e.g., cell) may be in suspension.

[0080] The fixation step of the biological material (e.g., the cell) can be carried out at a temperature between °C and 40 °C, or between 15 °C and 37 °C. For example, the fixation step of the biological material (e.g., the cell) can be carried out at a temperature between 20 °C and 27 °C, or between 34 °C and 40 °C. The fixation step of the biological material (e.g., the cell) can be carried out at a temperature of about 25 °C or about 37 °C. The fixation step of the biological material (e.g., the cell) can be carried out for between 30 minutes and 4 hours, or between 1 hour and 3 hours. The fixation step of the biological material (e.g., the cell) is preferably carried out for about 2 hours. The fixation step of the biological material (e.g., the cell) can be carried out for at least 30 minutes, at least 1 hour, at least 90 minutes, at least 2 hours, Petition 870250109009, dated 11 / 27 / 2025, page 57 / 249 49 / 190 at least 2.5 hours or at least 3 hours. The fixation step of the biological material (e.g., the cell) can be carried out in a humid chamber and / or in a CO2 incubator. The fixation step of the biological material (e.g., the cell) can be carried out under any conditions in which the cell does not dehydrate during the fixation step. That is, the fixation step of the biological material (e.g., the cell) ensures that the cell or elements of the biological material remain substantially immersed in liquid throughout the entire step.

[0081] In all embodiments described in this document, the nucleic acid may be or may comprise DNA and / or RNA, with DNA nucleic acid being preferred. The nucleic acid end(s) may be one or more DNA ends, one or more RNA ends, or one or more hybrid DNA / RNA ends, with the nucleic acid end(s) being one or more DNA ends being preferred.

[0082] The method described in this document is sufficiently sensitive and specific to detect the end(s) of single-stranded and double-stranded nucleic acid. Thus, the nucleic acid end(s) may be single-stranded end(s) and / or double-stranded end(s). The nucleic acid end(s) may be a single-stranded nucleic acid break or a Petition 870250109009, dated 11 / 27 / 2025, p. 58 / 249 50 / 190 double-stranded nucleic acid break. The nucleic acid end(s) may be a single-strand gap or a single-strand break. The nucleic acid end(s) may be a double-stranded blunt-end break or a double-strand break with a 3' protrusion.

[0083] The high sensitivity and / or specificity of the method also allows the detection of a single nucleic acid end in a cell. Thus, the nucleic acid end(s) can be a single nucleic acid end.

[0084] The methods described in this document are based on the addition of nucleic acid binding molecules to biological material under conditions such that the nucleic acid binding molecules bind to the nucleic acid end(s) on the biological material.

[0085] The nucleic acid-binding molecules used in the methods and kits described in this document may be any molecules capable of binding to the end of a nucleic acid molecule. The nucleic acid-binding molecules used in the methods and kits described in this document may also be bound by binding molecules (e.g., antibodies or fragments thereof) that may be bound (or bindable) to an oligonucleotide molecule. Preferably, a first part or site on the nucleic acid-binding molecules interacts Petition 870250109009, dated 11 / 27 / 2025, p. 59 / 249 51 / 190 with and / or binds to the nucleic acid end(s) and a second part or site on the nucleic acid binding molecules interacts with and / or is bound by (one of) the binding molecules. Nucleic acid binding molecules may be selected from a group comprising: (i) halogenated nucleotide or nucleoside molecules, such as BrdU, IdU, CldU, (ii) DNA precursor analogs, such as EdU (5-ethinyl2'-deoxyuridine), F-ara-EdU, 5-ethinyl-2'-deoxycytidine, (iii) biotinylated nucleotide molecules, (iv) ADP-ribose molecules, (v) protein molecules, (vi) nucleotide or nucleoside molecules labeled with markers; optionally wherein the markers are selected from a group comprising fluorescent molecules, or chemiluminescent molecules, or radioisotopes, or enzyme substrates, or biotin molecules.

[0086] Preferably, nucleic acid-binding molecules are halogenated nucleotide or nucleoside molecules, DNA precursor analogs and / or biotinylated nucleotide molecules.

[0087] Preferably, the nucleic acid-binding molecule(s) is / are not the same as the nucleic acid-binding protein. Petition 870250109009, dated 11 / 27 / 2025, page 60 / 249 52 / 190

[0088] Nucleic acid-binding molecules can be of the same type or of at least two, at least three, at least four, or at least five different types. For example, nucleic acid-binding molecules can be halogenated nucleotides of the same type (e.g., BrdU) or halogenated nucleotides of different types (e.g., BrdU and IdU). Nucleic acid-binding molecules can be halogenated nucleotide molecules and biotinylated nucleotide molecules. Nucleic acid-binding molecules can be biotinylated nucleotide molecules of different types (e.g., biotin-ATP, biotin-dGTP, biotin-dUTP, and / or biotin-dCTP).

[0089] Nucleic acid-binding molecules can bind to the nucleic acid end(s) by catalytic or non-catalytic means. Catalytic means may comprise enzymatic or non-enzymatic means. Nucleic acid-binding molecules can bind to the nucleic acid end(s) by an enzyme-catalyzed addition process, for example, using DNA polymerase I, terminal deoxynucleotide transferase (TdT), Klenow fragment, Phu polymerase, Taq polymerase, DNA polymerase T4, DNA polymerase T7, T4 polynucleotide kinase, RNA polymerases. Non-enzymatic means may comprise chemical factors, for example, chemical factors with catalytic properties. Non-enzymatic means Petition 870250109009, dated 11 / 27 / 2025, page 61 / 249 53 / 190 catalytic factors can include physical or biochemical factors.

[0090] In embodiments where the nucleic acid end(s) is / are a single-strand gap or a single-strand break, nucleic acid-binding molecules can bind to the nucleic acid end(s) with the aid of polymerase I. For example, in this embodiment, the nucleic acid-binding molecules can be biotinylated nucleotide molecules. In the context of this embodiment, proteinase treatment is performed for approximately 30 seconds.

[0091] In embodiments where the nucleic acid end(s) is / are a blunt-ended double-strand break or a double-strand break with a 3' protrusion, nucleic acid-binding molecules can bind to the nucleic acid end(s) with the aid of TdT (or a TdT reaction). For example, in this embodiment, the nucleic acid-binding molecules can be halogenated nucleotide or nucleoside molecules (e.g., BrdU). In the context of this embodiment, the proteinase treatment is performed for approximately 60 seconds.

[0092] A person skilled in the art would understand that, depending on the type of nucleic acid-binding molecule, the conditions under which the nucleic acid-binding molecules bind to the end(s) of the acid Petition 870250109009, dated 11 / 27 / 2025, p. 62 / 249 54 / 190 nucleic acids may differ. For example, if the nucleic acid-binding molecules are biotinylated nucleotide molecules, the conditions may require ideal conditions for a polymerase I reaction. For example, a temperature of about 37 °C. In this embodiment, the biological material may undergo a step of blocking endogenous biotin before the addition of biotinylated nucleotide molecules. If the nucleic acid-binding molecules are halogenated nucleotide or nucleoside molecules (e.g., BrdU), the conditions may require ideal conditions for a TdT reaction, for example, a temperature of about 37 °C. W.

[0093] The methods described in this document are based on the addition of two different binding molecules to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached (directly or indirectly, as defined in this document) to the nucleic acid at the nucleic acid end(s).

[0094] Each of the two different binding molecules may be or may comprise an antibody or fragments thereof, a streptavidin molecule, an avidin molecule, a streptavidin analogue, a Petition 870250109009, dated 11 / 27 / 2025, p. 63 / 249 55 / 190 biotin molecule, a peptide, a protein, a nucleic acid, an azide, or a polymer. The two different linking molecules may be of the same type or of different types. For example, the two different linking molecules may be or may comprise: (i) different antibodies or fragments thereof, such as a monoclonal antibody (or a fragment thereof) and a polyclonal antibody (or a fragment thereof); (ii) different antibodies or fragments thereof, such as a monoclonal antibody (or fragment thereof) specific for the nucleic acid-binding molecule used in the method, and a monoclonal antibody (or fragment thereof) specific for the nucleic acid-binding protein (i.e., the intrinsic nucleic acid-binding protein); (iii) an antibody or fragment thereof and streptavidin; (iv) an antibody or fragment thereof and an avidin; (v) an antibody or fragment thereof and a protein or peptide; or (vi) an antibody or fragment thereof and a biotin molecule.

[0095] Preferably, the two different binding molecules are (or comprise) two different monoclonal antibodies (or fragments thereof); one Petition 870250109009, dated 11 / 27 / 2025, p. 64 / 249 56 / 190 specific for the nucleic acid-binding molecule used in the method and the other specific for the nucleic acid-binding protein (i.e., the intrinsic nucleic acid-binding protein). Preferably, the two different binding molecules are (or comprise) two different monoclonal antibodies (or fragments thereof) from different species (e.g., a rabbit and a mouse).

[0096] The two different linking molecules can also be linked to one oligonucleotide molecule. For example, each of the two different linking molecules can be linked to an oligonucleotide molecule. Preferably, the oligonucleotide molecules of the two different linking molecules are different (for example, they have different sequences). Preferably, each of the two different linking molecules is linked to a different oligonucleotide molecule. Each oligonucleotide molecule can be covalently linked to each of the two different linking molecules.

[0097] The term different, as used in this document in the context of two different binding molecules, means that each of the two binding molecules binds to a different target molecule than the other binding molecule. Specifically, in methods, kits and Petition 870250109009, dated 11 / 27 / 2025, p. 65 / 249 57 / 190 uses described in this document, one of two different binding molecules binds to a nucleic acid-binding molecule and the other of the two different binding molecules binds to the nucleic acid-binding protein. The nucleic acid-binding protein and the nucleic acid-binding molecule are not the same molecules. The two different binding molecules may be molecules of the same type, for example, monoclonal antibodies (e.g., mouse monoclonal antibodies), but with specificity for different molecules. 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.

[0098] The two different linking molecules may comprise molecular probes. The molecular probes may be fluorophores and / or dyes. The two different linking molecules may comprise system components. Fluorogen Activating Protein (FAP) Protein) - Proximal Anchoring Activated Colorant (DAPA, Dye) Activated by Proximal Anchoring) (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 comprise a FAP and the other of the two Petition 870250109009, dated 11 / 27 / 2025, page 66 / 249 58 / 190 different binding molecules may comprise a HaloTag receptor. The two different binding molecules may additionally comprise antibodies that bind specifically to nucleic acid-binding molecules or to 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 the nucleic acid-binding protein, and the other of the different binding molecules may be a fusion protein of a FAP and an antibody against the nucleic acid-binding molecules. The two different binding molecules may comprise 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 additionally comprise antibodies that bind specifically to nucleic acid-binding molecules or to a nucleic acid-binding protein. Thus, for example, one of the different binding molecules may be a fusion protein of the N-terminal end of the Venus protein and an antibody against the nucleic acid-binding protein, and the other of the different binding molecules may be a fusion protein of the C-terminal end of the Venus protein and an antibody against the binding molecules. Petition 870250109009, dated 11 / 27 / 2025, p. 67 / 249 59 / 190 nucleic acid. Each of the two different linking molecules may comprise a nanobody, aptamer, or antibody linked to one of the two oligonucleotide molecules (i.e., one of the two different linking molecules may be linked to one of the two oligonucleotide molecules, and the other of the two different linking molecules may be linked to the other oligonucleotide molecule). The two oligonucleotide molecules may serve as a template for branched DNA signal amplification in a branched proximity hybridization assay. The two different linking molecules may comprise, respectively, a FRET donor probe and an acceptor probe.

[0099] A person skilled in the art would understand that, depending on the type of the two different binding molecules, the conditions under which the two different binding molecules bind to nucleic acid binding molecules and nucleic acid binding proteins may be different.

[00100] For example, if the two different binding molecules are monoclonal antibodies, the two different binding molecules can be incubated with the biological material for at least 30 minutes, preferably 60 minutes. The two different binding molecules can be incubated with the biological material at a temperature between 15 °C and 30 °C, preferably between 20 °C and 25 °C. Petition 870250109009, dated 11 / 27 / 2025, p. 68 / 249 60 / 190

[00101] The step of adding two different binding molecules to biological material may involve adding the two different binding molecules separately or together. Preferably, the two different binding molecules are added 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 the nucleic acid-binding protein (i.e., the intrinsic nucleic acid-binding protein) is added first, and the binding molecule that binds to nucleic acid-binding molecules is added second.

[00102] The step of adding two different binding molecules to biological material may include the following steps: i. Add one of two different binding molecules to the biological material. ii. Incubate one of the two different binding molecules with the biological material for at least 30 minutes, preferably 60 minutes. iii. Optionally, perform a washing step to remove any unbound molecules; iv. add the other of the two different binding molecules to the biological material; Petition 870250109009, dated 11 / 27 / 2025, p. 69 / 249 61 / 190 v. Incubate the other of the two different binding molecules with the biological material for at least 30 minutes, preferably 60 minutes; and vi. Optionally, perform a washing step to remove any unbound binding molecules.

[00103] The step of adding two different binding molecules to biological material may include the following steps: I. add one of two different binding molecules to the biological material, where the different binding molecule is specific to the nucleic acid-binding protein (i.e., the intrinsic nucleic acid-binding protein); ii. Incubate one of the two different binding molecules with the biological material for at least 30 minutes, preferably 60 minutes. iii. Optionally, perform a washing step to remove any unbound binding molecules. iv. Add the other of the two different binding molecules to the biological material, where the different binding molecule is specific for nucleic acid binding molecules. v. incubate the other of the two different binding molecules with the biological material for at least 30 minutes, preferably 60 minutes; and Petition 870250109009, dated 11 / 27 / 2025, p. 70 / 249 62 / 190 vi. Optionally, perform a washing step to remove any unbound molecules.

[00104] The methods described in this document are based on the presence of nucleic acid-binding proteins in biological material. Thus, the methods described in this document are based on the detection of intrinsic nucleic acid-binding proteins bound to nucleic acids at the nucleic acid end(s). More specifically, the methods described in this document are based on the detection of a colocalization of the intrinsic nucleic acid-binding protein and the nucleic acid end in biological material.

[00105] As explained in more detail in this document, nucleic acid-binding protein can be bound to the nucleic acid at the nucleic acid end(s), directly or indirectly. Those skilled in the art will understand that the detection method described in this document is based on the presence of a nucleic acid-binding protein bound to the nucleic acid at the nucleic acid end(s). For detection to work, the nucleic acid-binding protein must be sufficiently close to the nucleic acid at the nucleic acid end(s) so that detection of a colocalization of the nucleic acid-binding protein and the nucleic acid end via the binding of the nucleic acid-binding protein is possible. Petition 870250109009, dated 11 / 27 / 2025, p. 71 / 249 63 / 190 two different bonding molecules can occur.

[00106] Nucleic acid-binding protein may be bound to nucleic acid at the nucleic acid end(s) indirectly through interaction with an adapter protein. Nucleic acid-binding protein may be bound to nucleic acid at the nucleic acid end(s) indirectly as part of a protein complex. For example, the XRCC1 protein binds to nucleic acid indirectly through interactions with PARP1. For example, RAD51 and RPA bind to nucleic acid directly.

[00107] Thus, since nucleic acid-binding protein must have the ability to interact with or bind to nucleic acid (directly or indirectly), nucleic acid-binding protein can be a nucleic acid repair protein.

[00108] Nucleic acid-binding protein may be a protein involved in repairing nucleic acid breaks. Preferably, the nucleic acid-binding protein is bound to the nucleic acid end(s) (i.e., nucleic acid breaks) in biological material.

[00109] Nucleic acid-binding protein may be a protein involved in homologous recombination of nucleic acid (e.g., DNA) during double-strand break repair. Thus, nucleic acid-binding protein Petition 870250109009, dated 11 / 27 / 2025, page 72 / 249 64 / 190 could be RAD51.

[00110] Nucleic acid-binding protein can be a protein involved in the response to nucleic acid damage (e.g., DNA). Thus, the nucleic acid-binding protein could be replication protein A (RPA) (e.g., RPA70).

[00111] Nucleic acid-binding protein can be a protein involved in repairing errors in nucleic acid (e.g., DNA), for example, introduced during DNA replication. Thus, the nucleic acid-binding protein can be PMS2, PMS1, or MLH1.

[00112] Nucleic acid-binding protein may be p53, MSH2, ataxia-telangiectasia-related protein and Rad3, 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, RAD54-like DNA repair and recombination protein, PrimPol, REV1, terminal deoxynucleotidyl transferase, nude DNA polymerase, Fanconi anemia, complementation group C, FANCF, ERCC8, Artemis, ubiquitin ligase, RNF4, TP53BP1, AP endonuclease, ERCC4, transcription factor IIH, XRCC3, XRCC2, RecA, ERCC6, SLX4, Sirtuin 1, PTEN, replication protein A2, replication protein A3, Alkb3 homolog, alpha-ketoglutarate-dependent dioxygenase, exonuclease 5, Petition 870250109009, dated 11 / 27 / 2025, page 73 / 249 65 / 190 catalytic subunit of DNA polymerase alpha, cyclin H or PARP1 / 2.

[00113] The nucleic acid-binding protein may comprise 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 the phosphorylated form of H2AX (known as γH2AX).

[00114] Nucleic acid-binding protein can be a nucleic acid repair protein (e.g., DNA). Nucleic acid-binding proteins can be ATM, ATR, RPA, RAD51, MRE11, RAD17, RAD9A, RAD1, HUS1, TOPBP1, SMUG1, OGG1, PARP1, PARP2, PARP3, PARG, MGMT, TDP1, TDP2, MSH2, MSH3, MSH6, MLH1, PMS2, MSH4, MSH5, MSH3, PMS1, XPC, XPA, DDB1, DDB2, TFIIH, ERCC3, ERCC2, XRCC1, ERCC1, LIG1, RAD51B, RAD51D, 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.

[00115] Nucleic acid-binding protein can be a protein involved in replication. Nucleic acid-binding protein can be PCNA, RFC, RFA, Topo I, Topo II, polymerase a, polymerase d. Petition 870250109009, dated 11 / 27 / 2025, p. 74 / 249 66 / 190

[00116] Nucleic acid-binding protein may be a factor related to DNA repair, expressed by a gene: 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 (Espartano), 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, 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 (C19orf40), FAAP100, UBE2T (FANCT), XRCC6 (Ku70), XRCC5 (Ku80), PRKDC, LIG4, DUT, RRM2B (p53R2), PARK7 (DJ-1), Petition 870250109009, 27 / 11 / 2025, pág. 75 / 249 67 / 190 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 (DNase IV), 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, WDR48, HERC2, H2AX (H2AFX), CHAF1A (CAF1), SETMAR (METNASE), ATRX, BLM, RMI1, TOP3A, WRN, RECQL4, ATM, MPLKIP (TTDN1), RPA4, PRPF19 (PSO4), RECQL (RECQ1), ATR, ATRIP, MDC1, PCNA, RAD1, RAD9A, HUS1, RAD17 (RAD24), CHEK1, CHEK2, TP53, TP53BP1 (53BP1), RIF1, TOPBP1, CLK2 ou PER1.

[00117] In embodiments where the nucleic acid end(s) is / are a single-strand gap or a single-strand break, nucleic acid-binding molecules can bind to the nucleic acid end(s) by polymerase I catalysis. For example, in this embodiment, the nucleic acid-binding molecules can be biotinylated nucleotide molecules, and thus the nucleic acid end(s) is / are extended by the addition of nucleotide molecules. In this embodiment, for example, the nucleic acid-binding protein can be PMS2, PMS1, or MLH1. The two different binding molecules in this example can be Petition 870250109009, dated 11 / 27 / 2025, p. 76 / 249 68 / 190 a monoclonal anti-PMS2 (or anti-PMS1 or anti-MLH1) antibody and a monoclonal anti-biotin antibody. Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase for less than 60 seconds; b) adding biotinylated molecules and polymerase I to biological material under conditions such that the biotinylated molecules bind to the nucleic acid end(s) in the biological material; (c) add an anti-PMS2 (or anti-PMS1 or anti-MLH1) monoclonal antibody and an antibiotin monoclonal antibody to the biological material under conditions such that the anti-PMS2 monoclonal antibody binds to biotinylated molecules attached to the nucleic acid end(s) and the anti-PMS2 (or anti-PMS1 or anti-MLH1) monoclonal antibody binds to PMS2 (or PMS1 or MLH1) that is (indirectly) attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting a colocalization of PMS2 (or PMS1 or MLH1) and the nucleic acid end by binding of the two monoclonal antibodies.

[00118] In modalities in which the end(s) of Petition 870250109009, dated 11 / 27 / 2025, p. 77 / 249 69 / 190 nucleic acid is (are) a blunt end break of a double strand or a double strand break with a 3' protrusion, nucleic acid-binding molecules can bind to the nucleic acid end(s) by catalysis of Terminal Deoxynucleotide Transferase (TdT, or a TdT reaction, Terminal deoxynucleotide Transferase). For example, in this embodiment, the nucleic acid-binding molecules can be halogenated nucleotide or nucleoside molecules (e.g., BrdU) and thus the nucleic acid end(s) is / are extended by the addition of nucleotide molecules. In this embodiment, for example, the nucleic acid-binding protein can be RAD51 or RPA (e.g., RPA70). The two different binding molecules in this example can be an anti-RAD51 (or anti-RPA) monoclonal antibody and an anti-BrdU monoclonal antibody.Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase for less than 2 minutes; b) add BrdU and TdT to biological material under conditions such that BrdU binds to the nucleic acid end(s) in the biological material; c) Add an anti-RAD51 (or anti-RPA) monoclonal antibody and an anti-BrdU monoclonal antibody to the material. Petition 870250109009, dated 11 / 27 / 2025, p. 78 / 249 70 / 190 biological under conditions such that the anti-BrdU monoclonal antibody binds to BrdU attached to the nucleic acid end(s) and the anti-RAD51 (or anti-RPA) monoclonal antibody binds to RAD51 (or RPA) that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting a colocalization of RAD51 (or RPA) and the nucleic acid end by binding the two monoclonal antibodies.

[00119] The method may further comprise a step of adding additional linking molecules that bind to the two different linking molecules after the step of adding the two different linking molecules. In this embodiment, the two different linking molecules may not be linked to oligonucleotide molecules. In this embodiment, the additional linking molecules may be linked to an oligonucleotide molecule, and thus the additional linking molecules connect the oligonucleotide molecules to the two different linking molecules. Preferably, the additional linking molecules are two different additional linking molecules. In the method described in this document, each of the two different additional linking molecules may be linked to a different oligonucleotide molecule (i.e., an oligonucleotide molecule of a different sequence). Petition 870250109009, dated 11 / 27 / 2025, p. 79 / 249 71 / 190

[00120] Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different binding molecules to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); d) Add two different additional binding molecules to the biological material, where one of the two different additional binding molecules binds to the binding molecule attached to the nucleic acid binding molecules, and the other of the two different additional binding molecules binds to the binding molecules attached to the binding protein. Petition 870250109009, dated 11 / 27 / 2025, p. 80 / 249 72 / 190 nucleic acid; e) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules and two additional binding molecules.

[00121] As used in this document, the term “different,” in the context of two additional binding molecules, means that each of the two additional binding molecules binds to one, but not the other, of the binding molecules. Thus, each of the different additional molecules may be specific for a different binding molecule. Additional binding molecules may be of the same type (e.g., binding monoclonal antibodies, each specific for a different binding molecule) or of different types. Preferably, the two different additional binding molecules are monoclonal antibodies; one is an antibody that binds to the binding molecule attached to nucleic acid-binding molecules attached to the nucleic acid end(s), and the other is an antibody that binds to the binding molecule attached to the nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s).

[00122] Additional binding molecules may comprise an antibody or a fragment thereof, a Petition 870250109009, dated 11 / 27 / 2025, p. 81 / 249 73 / 190 streptavidin molecule, an avidin molecule, a biotin molecule, a protein, a peptide, a nucleic acid, an azide, or a polymer. Additional binding molecules may comprise at least two different additional binding molecules (e.g., two different antibodies, each specific for one of the two different binding molecules). For example, the two different binding molecules that bind to the nucleic acid-binding molecule and the nucleic acid-binding protein, respectively, may be primary antibodies or fragments thereof, and the additional binding molecules may be secondary antibodies or fragments thereof that specifically target the primary antibodies.For example, the two different linking molecules that bind to the nucleic acid-binding molecule may be primary mouse and rabbit antibodies, and the additional linking molecules may be secondary anti-mouse and anti-rabbit antibodies. Additional linking molecules may be directly linked (e.g., conjugated) to an oligonucleotide molecule. Preferably, each of the two additional linking molecules is directly linked (e.g., conjugated) to a different oligonucleotide molecule (i.e., an oligonucleotide molecule with a different sequence).

[00123] The method may additionally include, Petition 870250109009, dated 11 / 27 / 2025, p. 82 / 249 74 / 190 after the step of adding two different linking molecules (or after the step of adding the additional linking molecules), a step of adding additional oligonucleotide molecules that hybridize with the oligonucleotide molecules linked to the two different linking molecules (or to the additional linking molecules) to form a circular amplification pattern.

[00124] The additional oligonucleotide molecules may have the same sequence or at least two different sequences. The additional oligonucleotide molecules may hybridize with two oligonucleotide molecules linked to two different linking molecules (or to additional linking molecules) to form a circular template. The two additional oligonucleotide molecules may be linked together to form a circular template. Thus, the method may further comprise a step of linking two additional oligonucleotide molecules. The method may further comprise a step of extending the two additional oligonucleotide molecules followed by a linking step. Preferably, the additional oligonucleotide molecules have at least two different sequences. The circular template may comprise or consist of the two additional oligonucleotide molecules.The circular model may additionally comprise an extension product of the two oligonucleotide molecules. Petition 870250109009, dated 11 / 27 / 2025, page 83 / 249 75 / 190 additional. The circular model is suitable for rolling circle amplification. The circular amplification model can be formed by linking the additional oligonucleotide molecules (or the two different additional oligonucleotide molecules).

[00125] The method may further comprise a nucleic acid amplification step to produce an amplification product prior to the nucleic acid end detection step. Thus, nucleic acid end detection may comprise a nucleic acid amplification step to produce an amplification product that is detected. The nucleic acid amplification may be a rolling circle amplification.

[00126] Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid binding molecules to biological material under conditions such that the nucleic acid binding molecules bind to the end(s) of Petition 870250109009, dated 11 / 27 / 2025, p. 84 / 249 76 / 190 nucleic acid in biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-linked molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-linking protein that is attached to the nucleic acid at the nucleic acid end(s), wherein each of the two different linking molecules is attached to an oligonucleotide molecule; d) add additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different linking molecules to form a circular amplification template; (e) perform nucleic acid amplification to produce an amplification product; and f) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00127] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the Petition 870250109009, dated 11 / 27 / 2025, p. 85 / 249 77 / 190 biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-linked molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-linking protein that is attached to the nucleic acid at the nucleic acid end(s), wherein each of the two different linking molecules is attached to a different oligonucleotide molecule; d) add additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different linking molecules to form a circular amplification template; (e) perform nucleic acid amplification to produce an amplification product; and f) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00128] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: Petition 870250109009, dated 11 / 27 / 2025, p. 86 / 249 78 / 190 (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-linked molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-linking protein that is attached to the nucleic acid at the nucleic acid end(s), wherein each of the two different linking molecules is attached to a different oligonucleotide molecule; d) add two different additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different linker molecules to form a circular amplification template; (e) perform nucleic acid amplification to produce an amplification product; and f) detect the amplification product to detect Petition 870250109009, dated 11 / 27 / 2025, p. 87 / 249 79 / 190 the nucleic acid end(s) in biological material.

[00129] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-linked molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-linking protein that is attached to the nucleic acid at the nucleic acid end(s), wherein each of the two different linking molecules is attached to an oligonucleotide molecule; d) adding additional oligonucleotide molecules that hybridize with the attached oligonucleotide molecules Petition 870250109009, dated 11 / 27 / 2025, p. 88 / 249 80 / 190 to the two different binding molecules to form a circular amplification template; (e) perform nucleic acid amplification to produce an amplification product; and f) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00130] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different binding molecules to the biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the end(s) of the nucleic acid and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the Petition 870250109009, dated 11 / 27 / 2025, p. 89 / 249 81 / 190 nucleic acid end(s), wherein each of the two different linker molecules is attached to a different oligonucleotide molecule; d) add additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different linking molecules to form a circular amplification template; (e) perform nucleic acid amplification to produce an amplification product; and f) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00131] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (approximately 7 pg / mL) for less than 2 minutes at a temperature of approximately 22 °C; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different binding molecules to biological material under conditions such that one of the molecules Petition 870250109009, dated 11 / 27 / 2025, p. 90 / 249 82 / 190 of the linker binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linker molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s), wherein each of the two different linker molecules is attached to a different oligonucleotide molecule; d) add two different additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different linker molecules to form a circular amplification template; (e) perform nucleic acid amplification to produce an amplification product; and f) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00132] Thus, the method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the molecules of Petition 870250109009, dated 11 / 27 / 2025, p. 91 / 249 83 / 190 nucleic acid binding binds to the nucleic acid end(s) in biological material; (c) adding two different binding molecules to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s), (d) adding two different additional linking molecules to the biological material, wherein one of the two different additional linking molecules binds to the nucleic acid-binding linking molecule, and the other of the two different additional linking molecules binds to the nucleic acid-binding protein-binding linking molecules; wherein each of the two different additional linking molecules is linked to an oligonucleotide molecule; (e) Add additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different additional linker molecules to form a circular amplification template; f) perform nucleic acid amplification to produce an amplification product; and g) detect the amplification product to detect Petition 870250109009, dated 11 / 27 / 2025, p. 92 / 249 84 / 190 the nucleic acid end(s) in biological material.

[00133] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different binding molecules to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s), d) Add two different additional linking molecules to the biological material, where one of the two different additional linking molecules binds to the linking molecule attached to nucleic acid linking molecules, and the other of the two different additional linking molecules Petition 870250109009, dated 11 / 27 / 2025, p. 93 / 249 85 / 190 binds to nucleic acid-binding protein-linked binding molecules; wherein each of the two different additional binding molecules is linked to a different oligonucleotide molecule; (e) Add additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different additional linker molecules to form a circular amplification template; f) perform nucleic acid amplification to produce an amplification product; and g) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00134] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) add two different bonding molecules to Petition 870250109009, dated 11 / 27 / 2025, p. 94 / 249 86 / 190 biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s), (d) adding two different additional linking molecules to the biological material, wherein one of the two different additional linking molecules binds to the linking molecule attached to nucleic acid-binding molecules, and the other of the two different additional linking molecules binds to the linking molecules attached to nucleic acid-binding protein; wherein each of the two different additional linking molecules is attached to a different oligonucleotide molecule; (e) Add two different additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different additional linker molecules to form a circular amplification template; f) perform nucleic acid amplification to produce an amplification product; and g) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00135] The method for end detection(s) of Petition 870250109009, dated 11 / 27 / 2025, p. 95 / 249 87 / 190 nucleic acid in a biological material containing nucleic acids may involve the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (approximately 7 pg / mL) for less than 2 minutes at a temperature of approximately 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different binding molecules to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s), d) Add two different additional binding molecules to the biological material, where one of the two different additional binding molecules binds to the binding molecule attached to the nucleic acid binding molecules, and the other of the two different additional binding molecules binds to the binding molecules attached to the binding protein. Petition 870250109009, dated 11 / 27 / 2025, p. 96 / 249 88 / 190 nucleic acid; wherein each of the two different additional linker molecules is attached to an oligonucleotide molecule; (e) Add additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different additional linker molecules to form a circular amplification template; f) perform nucleic acid amplification to produce an amplification product; and g) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00136] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (approximately 7 pg / mL) for less than 2 minutes at a temperature of approximately 22°C; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different binding molecules to biological material under conditions such that one of the molecules Petition 870250109009, dated 11 / 27 / 2025, p. 97 / 249 89 / 190 of the binding molecules attach to nucleic acid-binding molecules attached to the nucleic acid end(s), and the other binding molecule attaches to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s). (d) adding two different additional linking molecules to the biological material, wherein one of the two different additional linking molecules binds to the linking molecule attached to nucleic acid-binding molecules, and the other of the two different additional linking molecules binds to the linking molecules attached to nucleic acid-binding protein; wherein each of the two different additional linking molecules is attached to a different oligonucleotide molecule; (e) Add additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different additional linker molecules to form a circular amplification template; f) perform nucleic acid amplification to produce an amplification product; and g) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00137] The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: Petition 870250109009, dated 11 / 27 / 2025, p. 98 / 249 90 / 190 a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase K at a concentration of 6 pg / mL to 8 pg / mL (e.g., about 7 pg / mL) for less than 2 minutes at a temperature of about 22 °C (i.e., room temperature); b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different binding molecules to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s), (d) adding two different additional linking molecules to the biological material, wherein one of the two different additional linking molecules binds to the linking molecule attached to nucleic acid-binding molecules, and the other of the two different additional linking molecules binds to the linking molecules attached to nucleic acid-binding protein; wherein each of the two different additional linking molecules is attached to a molecule of Petition 870250109009, dated 11 / 27 / 2025, p. 99 / 249 91 / 190 different oligonucleotide; (e) Add two different additional oligonucleotide molecules that hybridize with the oligonucleotide molecules attached to the two different additional linker molecules to form a circular amplification template; f) perform nucleic acid amplification to produce an amplification product; and g) detect the amplification product to detect the nucleic acid end(s) in the biological material.

[00138] In the methods described in this document, the detection of the colocalization of the nucleic acid-binding protein and the nucleic acid end may comprise the detection of the amplification product, a proximity binding assay, or a proximity-induced reaction with fluorophores or dyes. The detection of the co-localization of the nucleic acid-binding protein and the nucleic acid end may comprise the detection of a branched probe or a FRET probe.

[00139] The step of detecting the nucleic acid end(s) in biological material (e.g., detection of the amplification product) can be performed by means of microscopy, automated large-cell analysis methods, spectroscopy, fluorescence microscopy (wide-field, confocal, multifocal, of Petition 870250109009, dated 11 / 27 / 2025, pp. 100 / 249 92 / 190 super-resolution, catapult-fed, laser scanning, high-throughput, high-content), fluorimetry, transmitted light optical microscopy for absorption detection, flow cytometry, cell separation (FACS) and / or mass spectrometry. The detection step of the nucleic acid end(s) in the biological material (e.g., detection of the amplification product) may include the addition of a detection molecule, for example, a labeled molecule capable of recognizing and binding to the amplification product or to two additional binding molecules. For example, the labeled molecule may bind to the sequence of the additional oligonucleotide molecule. The labeled molecule may comprise a nucleic acid. The labeled molecule may comprise a barcode or a label.

[00140] Detection of the colocalization of the nucleic acid-binding protein and the nucleic acid end can be performed by photoactivation of the binding molecules described in this document, or by photoactivation of probes linked to the amplification product. Detection of the colocalization of the nucleic acid-binding protein and the nucleic acid end can be performed by immunocytochemical analysis using an antibody (e.g., polyclonal serum) against GFP that recognizes both fragments of a split protein. Detection of the co-localization of Petition 870250109009, dated 11 / 27 / 2025, p. 101 / 249 Co-localization of nucleic acid-binding protein and nucleic acid end can be performed by fluorescence analysis of activated fluorophores or by fluorescence analysis of probes or labels attached to the amplification product. Detection of co-localization of nucleic acid-binding protein and nucleic acid end can be performed by detection of the amplification product (e.g., using a label, for example, a fluorescent label). Detection of co-localization of nucleic acid-binding protein and nucleic acid end can be performed by proximity-induced SNAr reactions of lysine-linked fluorophores (Hymel et al., 2014, “Detection of Protein-Protein Interactions by Proximity-Oriented SNAr Reactions of Lysine-Linked Fluorophores”). Detection of co-localization of nucleic acid-binding protein and nucleic acid end can be performed by detection of FRET probes.Thus, one of two different binding molecules (i.e., the donor probe) can be excited by an external light source and transfer its energy to the other different binding molecule (i.e., the acceptor probe). The excited acceptor probe can emit light of a different (usually longer) wavelength, which can be detected and measured. Detection of the co-localization of the nucleic acid-binding protein and the nucleic acid end can be achieved. Petition 870250109009, dated 11 / 27 / 2025, p. 102 / 249 94 / 190 performed by a branched proximity hybridization assay to detect branched probes (e.g., DNA probes).

[00141] Preferably, in the methods described in this document, detection is performed in situ.

[00142] The methods described in this document may comprise one or more additional steps prior to the incubation of the biological material with a proteinase. The method may comprise a step of permeabilizing the biological material (e.g., a cell). The method may comprise an additional step of increasing the accessibility of the nucleic acid end(s). The method may comprise a step of blocking nonspecific binding site(s) for at least one of the molecules used in the method. The method may comprise a step of modifying the nucleic acid (DNA) ends in the biological material.The permeabilization step of biological material (e.g., a cell) may include chemical and / or physical procedures that compromise the integrity of the biological material (e.g., the cell) in order to allow or facilitate access by the molecules used in the method to the nucleic acid and nucleic acid-binding protein in the biological material (e.g., the cell).

[00143] The additional step of increasing the accessibility of the nucleic acid end(s) may Petition 870250109009, dated 11 / 27 / 2025, page 103 / 249 95 / 190 can be performed using the methods described in this document in addition to the step of incubating the biological material with a proteinase. The additional step of increasing the accessibility of the nucleic acid end(s) can further facilitate the access of the molecules used in the methods to the nucleic acid end(s) in the biological material (e.g., in the cell).

[00144] The nonspecific binding site blocking step for at least one of the molecules used in the method may involve blocking all nonspecific binding sites of the molecules used in the method. The term “blocking” means a procedure that prevents the binding of critical molecules used in the assay, for example, antibodies, to molecular targets other than those for which the selected molecules exhibit useful affinity in the assay. A typical example of blocking is a procedure used in the detection of antibodies (immunodetection) of a molecule of interest in biological material. The antibody is directed against a well-defined antigen, but it can also bind to various cellular components through weak chemical interactions. This nonspecific binding is minimized by the prior addition of blocking agents (such as albumin) that occupy such binding sites.Therefore, the methods described in this document may additionally include one more step. Petition 870250109009, dated 11 / 27 / 2025, page 104 / 249 96 / 190 blockage of one or more nonspecific binding sites of molecules after the step of adding two different binding molecules to the biological material and / or the step of adding additional binding molecules.

[00145] The nucleic acid (DNA) end modification step in biological material can be performed after the proteinase treatment step and before the addition of nucleic acid-binding molecules. The nucleic acid end modification step can be performed by chemical or physical means. For example, nucleic acid end modification can be performed using T4 polynucleotide kinase.

[00146] The methods described in this document may comprise one or more washing steps. For example, the proteinase and / or proteinase inhibitor may be removed by washing after the incubation step of the biological material with the proteinase. The washing step may be performed after the addition step of nucleic acid-binding molecules. The washing step may be performed after the addition step of two different binding molecules. The washing step may be performed after the addition step of additional oligonucleotide molecules. The washing step may be performed after the amplification step. The washing step removes unbound material, which may result in a sharper detection signal and / or less background noise. Petition 870250109009, dated 11 / 27 / 2025, page 105 / 249 97 / 190

[00147] Figure 1 shows an example of the invention method after the proteinase treatment step. In step 1, a single-strand break is created in the nucleic acid (e.g., DNA). The intrinsic nucleic acid-binding protein (e.g., PMS2, PMS1, or MLH1) binds to the nucleic acid at the end of the nucleic acid to, for example, repair the break created. In step 2, nucleic acid-binding molecules bind to the end of the nucleic acid. This can be accomplished with the aid of an enzyme, for example, polymerase I (indicated with a gray circle in step 2). This reaction generates a chain of nucleic acid-binding molecules (visualized in step 3). In step 3, two different binding molecules (linked to oligonucleotide molecules) bind, respectively, to the nucleic acid-binding protein and to the nucleic acid-binding molecules.In step 4, additional oligonucleotide molecules hybridize with the oligonucleotide molecules attached to the two different linking molecules. In step 5, the additional oligonucleotide molecules link together to form a circular template. In step 6, rolling circle amplification is performed on the circular template to produce an amplified product. In step 7, detection molecules (e.g., fluorescent probes) bind to the amplification product, allowing detection of the amplified product. Petition 870250109009, dated 11 / 27 / 2025, page 106 / 249 98 / 190

[00148] Figure 2 shows another example of the method of the invention. In this case, the steps of the method are the same as in Figure 1, except that additional linking molecules (which are linked to the oligonucleotide molecules) link to two different linking molecules (which, in this example, are not linked to the oligonucleotide molecules).

[00149] Figure 3 shows another example of the method of the invention. The steps are the same as in Figure 1. However, in this case, the nucleic acid-binding molecules (e.g., nucleotide analogs) are incorporated directly into the single-strand break of the nucleic acid (e.g., DNA). Thus, in this example, a chain of nucleic acid-binding molecules is not formed.

[00150] Figure 4 shows another example of the invention's method. In this example, the nucleic acid break is a double-strand break. The intrinsic nucleic acid-binding protein (e.g., Rad51 or RPA) binds to the nucleic acid at the nucleic acid end to, for example, repair the break created. In step 2, nucleic acid-binding molecules bind to the nucleic acid end. This can be done with the aid of an enzyme, for example, TdT (indicated with a gray circle in step 2). This reaction generates a chain of nucleic acid-binding molecules (visualized in step 3). In step 4, two molecules of Petition 870250109009, dated 11 / 27 / 2025, page 107 / 249 In step 5 (which is optional), additional oligonucleotide-linked binding molecules bind to the two different binding molecules. In step 6, additional oligonucleotide molecules hybridize with the oligonucleotide molecules linked to the additional binding molecules and are linked together to form a circular template. In step 7, rolling circle amplification is performed on the circular template to produce an amplified product. In step 8, detection molecules (e.g., fluorescent probes) bind to the amplification product, allowing detection of the amplified product.

[00151] The method described in this document allows for the direct detection and visualization of nucleic acid end(s). The method described in this document allows for marking the presence and position of a single-strand break in nucleic acid. Thus, the method is particularly useful for detecting damage in nucleic acids, which has wide applicability. For example, the method can be used to assess the quality of DNA in a given individual. The method can also be used to study the mechanism of induction, signaling, and repair of DNA damage. In the context of cancer cells, the method can be particularly useful for evaluating the effectiveness of a therapeutic agent. Petition 870250109009, dated 11 / 27 / 2025, page 108 / 249 100 / 190

[00152] Thus, in one aspect, the invention provides a method for evaluating the efficacy of a therapeutic agent, the method comprising the steps of: a) perform the detection method as described in this document on a sample obtained from an individual before administration of the therapeutic agent, b) perform the detection method as described in this document on a sample obtained from the individual after administration of the therapeutic agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the therapeutic agent is effective if the amount of nucleic acid end(s) detected is greater in step a) than in step b), where steps a) and b) can be performed in any order.

[00153] The individual may be an animal; preferably, the individual is human.

[00154] Preferably, the nucleic acid end(s) are DNA ends. Even more preferably, the nucleic acid end(s) are single-stranded nucleic acid ends (e.g., DNA).

[00155] The therapeutic agent may be an oncology drug or a drug candidate. The method may be Petition 870250109009, dated 11 / 27 / 2025, p. 109 / 249 101 / 190 is used to evaluate the therapeutic efficacy of a drug candidate. The therapeutic agent may target a protein involved in the base mismatch repair (MMR) pathway. Thus, the therapeutic agent may target PMS1, PMS2, and / or MLH1. Therefore, in the detection methods described in this document, the nucleic acid-binding protein may be PMS1, PMS2, or MLH1. The therapeutic agent may inhibit a protein involved in the base mismatch repair pathway. In this way, once inhibited, the protein will not be able to bind to the nucleic acid end(s), and therefore, the detection method will show a reduced number of nucleic acid ends detected in the biological material.

[00156] The sample can be obtained from the individual 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 the nucleic acid end-finding detection method at multiple time points after administration of the therapeutic agent. This approach allows for the evaluation of the long-term effect of a therapeutic agent.

[00157] In one aspect, the invention provides a method for evaluating the effectiveness of a therapeutic agent, the method comprising the steps of: Petition 870250109009, dated 11 / 27 / 2025, page 110 / 249 102 / 190 a) perform the detection method as described in this document on a sample obtained from an individual before administration of the therapeutic agent, b) perform the detection method as described in this document on a sample obtained from the individual after administration of the therapeutic agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the therapeutic agent is effective if the amount of nucleic acid end(s) detected is greater in step b) than in step a), where steps a) and b) can be performed in any order.

[00158] The individual may be an animal; preferably, the individual is human.

[00159] Preferably, the nucleic acid end(s) are DNA ends. Even more preferably, the nucleic acid end(s) are double-stranded nucleic acid ends (e.g., DNA).

[00160] The therapeutic agent may be an oncology drug or a drug candidate. The method can be used to evaluate the therapeutic efficacy of a drug candidate. The therapeutic agent may target a protein involved in nucleic acid error repair. Petition 870250109009, dated 11 / 27 / 2025, page 111 / 249 103 / 190 (e.g., DNA) or a protein involved in homologous nucleic acid recombination (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.

[00161] The sample can be obtained from the individual 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 the nucleic acid end-finding detection method at multiple time points after administration of the therapeutic agent. This approach allows for the evaluation of the long-term effect of a therapeutic agent.

[00162] The sample can be obtained from a tumor biopsy. The sample may contain a cancerous cell.

[00163] The method described in this document may also be useful for assessing nucleic acid damage caused by an agent (e.g., a chemical or environmental agent).

[00164] Thus, in one aspect, the invention relates to a method for evaluating nucleic acid damage caused by an agent, the method comprising the steps of: a) perform the detection method as described in this document on a sample obtained from an individual before Petition 870250109009, dated 11 / 27 / 2025, page 112 / 249 104 / 190 of the administration of, or exposure to, an agent, b) perform the detection method as described in this document on a sample obtained from the individual after administration of, or exposure to, the agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the agent causes damage to the nucleic acid if the amount of nucleic acid end(s) detected is greater in step b) than in step a), where steps a) and b) can be performed in any order.

[00165] Nucleic acid damage can be a single-strand break or a double-strand break. Preferably, the nucleic acid end(s) are DNA ends. Thus, nucleic acid damage can be a single-strand DNA break or a double-strand DNA break. In the context of single-strand breaks, preferably, the method is based on the detection of PMS1, PMS2, or MLH1 as nucleic acid-binding proteins. In the context of double-strand breaks, preferably, the method is based on the detection of Rad51 or RPA as nucleic acid-binding proteins.

[00166] The agent can be a therapeutic agent, a chemical agent, or an environmental agent.

[00167] In the context of a therapeutic agent, the Petition 870250109009, dated 11 / 27 / 2025, p. 113 / 249 The method described in this document is capable of detecting nucleic acid damage caused by a therapeutic agent, which may constitute a side effect. For example, the therapeutic agent may have a therapeutic effect against a specific disease or disorder and, at the same time, cause nucleic acid damage in certain cell types. Thus, the method described in this document is capable of identifying whether the therapeutic agent causes nucleic acid damage (e.g., DNA).

[00168] The chemical agent may be a toxin, ink, adhesive, paint, oil, lubricant, hair dye, laboratory reagent, welding fumes, hazardous medicinal product or cleaning fluid.

[00169] The environmental agent can be a pollutant, a bacterium (or part thereof), a virus (or part thereof), or a radioactive substance. The pollutant can be an air pollutant (e.g., particulate matter, ozone, nitrogen dioxide, carbon monoxide, or sulfur dioxide); a water pollutant (e.g., bacteria, viruses, parasites, fertilizers, pesticides, pharmaceuticals, nitrates, phosphates, plastics, or fecal matter); or a soil pollutant (e.g., pesticides, petroleum products, radon, asbestos, lead, chromated copper arsenate, or creosote).

[00170] The term administration, as used Petition 870250109009, dated 11 / 27 / 2025, pp. 114 / 249 106 / 190 in this document refers to any type of administration of an agent to an individual. For example, the agent may be administered to the individual by any acceptable route of administration, including, but not limited to, oral, topical (including transdermal), and parenteral routes.

[00171] The term “exposure to” an agent, as used in this document, is intended to encompass any type of exposure. For example, an individual may be exposed to the agent by inhalation from the air, ingestion, absorption through the skin, eyes or a body membrane (e.g., nasal or vaginal membrane), or by direct contact with fluids containing the agent (e.g., in the case of injury).

[00172] The sample can be obtained from the individual after 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 or exposure to the agent. The method may include performing the nucleic acid end-of-life detection method at multiple time points after administration or exposure to the agent. This approach allows for the assessment of the long-term effect of the agent on the individual's health status.

[00173] The methods described in this document are preferably performed in vitro.

[00174] The invention provides a method for forecasting Petition 870250109009, dated 11 / 27 / 2025, page 115 / 249 107 / 190 the response of an individual diagnosed with cancer to an anticancer treatment, comprising (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual by performing the detection method as described in this document and (ii) predicting the individual's response to an anticancer treatment based on the determined level of the nucleic acid-binding protein in the sample.

[00175] The invention provides a method for selecting a personalized therapy for an individual diagnosed with cancer, comprising (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual by performing the detection method as described in this document; and (ii) selecting a personalized therapy for the individual based on the determined level of the nucleic acid-binding protein in the sample.

[00176] The invention provides a method for classifying an individual diagnosed with cancer into a cohort of patients, comprising (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual by performing the detection method as described in this document; and (ii) classifying an individual diagnosed with cancer into a cohort of patients based on the determined level of the nucleic acid-binding protein in the sample. Petition 870250109009, dated 11 / 27 / 2025, page 116 / 249 108 / 190

[00177] The invention provides a method for predicting whether a tumor in an individual diagnosed with cancer is capable of repairing DNA by homologous recombination, comprising (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual performing the detection method as described in this document; and (ii) predicting whether a tumor in the individual diagnosed with cancer is capable of repairing DNA by homologous recombination based on the determined level of the nucleic acid-binding protein in the sample.

[00178] The invention provides a method for evaluating the state of a mismatch repair pathway in a sample comprising a tumor cell obtained from an individual, the method comprising (i) determining the level of a nucleic acid-binding protein in the tumor cell by performing the detection method as described in this document; and (ii) evaluating the state of the mismatch repair pathway based on the determined level of the nucleic acid-binding protein in the tumor cell.

[00179] In all embodiments described in this document, preferably the nucleic acid-binding protein is RAD51, RPA (e.g., RPA70), PMS1, PMS2, or MLH1.

[00180] In all embodiments described in this document, preferably, the sample contains cells Petition 870250109009, dated 11 / 27 / 2025, page 117 / 249 109 / 190 tumors.

[00181] The antineoplastic treatment or agent may be a poly(ADP-ribose) polymerase (PARP) inhibitor. PARP is a protein (enzyme) that helps damaged cells repair themselves. As a cancer treatment, PARP inhibitors prevent PARP from performing its repair function in cancer cells, leading to their death. Normally, the BRCA1 and BRCA2 genes play a role in cell repair in the body. Cells are less likely to repair themselves when there is a mutation in one or both of these genes. Patients with mutations in the BRCA genes have an increased risk of certain types of cancer, including breast, ovarian, and prostate cancer. Cancer cells with mutations in the BRCA genes already have a deficient repair system. Thus, blocking PARP with a PARP inhibitor drug prevents these cells from repairing themselves, leading to their death.

[00182] In all modalities described in this document, preferably, the individual should not have received chemotherapy or an antineoplastic agent at least 24 hours prior to sample collection. In all modalities described in this document, preferably, the sample should not have been exposed to an agent or environment that induces DNA damage prior to the step of determining the level of nucleic acid-binding protein in the sample.

[00183] In all the modalities described in this Petition 870250109009, dated 11 / 27 / 2025, pp. 118 / 249 110 / 190 document, preferably, the step of determining the level of nucleic acid-binding protein in a sample comprises detecting or visualizing the location and / or distribution of the nucleic acid-binding protein in the sample (e.g., in a cell, such as a tumor cell). The step of determining the level of a nucleic acid-binding protein in a sample may comprise determining the level of cells with that protein in the sample. Preferably, the cells are tumor cells.

[00184] In all embodiments described in this document, preferably, the determined level of nucleic acid-binding protein is compared to a reference value. If the nucleic acid-binding protein level in the sample is lower than the reference value, this indicates that (i) the individual is expected to respond to antineoplastic treatment, (ii) the therapy to be selected comprises specific agents to treat tumors with deficiencies in response to DNA damage, (iii) the individual is classified in a cohort distinguished by responding to antineoplastic treatment, or (iv) the tumor is capable of performing DNA repair by homologous recombination. If the nucleic acid-binding protein level in the sample is higher than the reference value, this indicates that (i) the individual is expected not to respond to treatment. Petition 870250109009, dated 11 / 27 / 2025, pp. 119 / 249 111 / 190 antineoplastic, (ii) the therapy to be selected does not comprise agents specific to treat tumors with deficiencies in response to DNA damage, (iii) the individual is classified in a cohort of patients distinguished by not responding to antineoplastic treatment, or (iv) the tumor is not capable of performing DNA repair by homologous recombination.

[00185] The term “reference value” refers to a laboratory value used as a reference for values / data obtained from a sample taken from an individual. A reference value may be based on a value obtained from a group of patients considered representative. Alternatively, a reference value may be based on an individual sample, such as a value obtained from a patient sample at an earlier time point. For example, a reference value may come from a sample collected from a patient without a specific mutation affecting the response to antineoplastic treatment.

[00186] The expression “tumors with DNA damage response deficiencies” refers to tumors or tumor cells with an altered DNA damage response pathway. This may include abnormal expression of a protein involved in the DNA damage response. The protein may be ATM, ATR, RPA, RAD51, MRE11, RAD17, RAD9A, RAD1, HUS1, Petition 870250109009, dated 11 / 27 / 2025, pp. 120 / 249 112 / 190 TOPBP1, SMUG1, OGG1, PARP1, PARP2, PARP3, PARG, MGMT, TDP1, TDP2, MSH2, MSH3, MSH6, MLH1, PMS2, MSH4, MSH5, MSH3, PMS1, XPC, DNPH1, POLA, POLB, POLD, POLE, REV3L, POLQ, FEN1, TREX1, TREX2, EXO1, APTX1, HERC2, RNF8, RNF4, H2AX, BLM, WRN, RECQL4, ATRIP, PCNA, TP53, RIF1, TOPBP2. Alterations in the DNA damage response pathway can lead to genomic instability.

[00187] The state of the mismatch repair pathway may indicate that one or more of the proteins involved in mismatch repair are deficient or functioning abnormally. Alternatively, the state of the mismatch repair pathway may indicate that the tumor cell possesses a fully functional mismatch repair pathway.

[00188] The invention also provides a medicament for use in the treatment of cancer in an individual, wherein the individual has been identified as a responder to said medicament by one of the methods described in this document.

[00189] The methods described in this document can be performed using the kits described in this document. A person skilled in the art would understand that all molecules, reagents, and approaches described in the context of Petition 870250109009, dated 11 / 27 / 2025, pp. 121 / 249 113 / 190 methods are equally applicable to aspects of the invention relating to the kit. However, certain preferred embodiments are also highlighted in the context of aspects of the invention relating to the kit.

[00190] The invention provides a kit for detecting nucleic acid end(s) in a biological material, the kit comprising: a) nucleic acid-binding molecules; b) a linker molecule that binds to nucleic acid-binding molecules; and c) a binding molecule that binds to a nucleic acid-binding protein.

[00191] The invention also provides the use of a kit for detecting nucleic acid end(s) in biological material, the kit comprising: a) nucleic acid-binding molecules; b) a linker molecule that binds to nucleic acid-binding molecules; and c) a binding molecule that binds to a nucleic acid-binding protein.

[00192] The kit may contain a proteinase, preferably proteinase K.

[00193] The kit for detecting nucleic acid end(s) in a biological material may comprise: a) nucleic acid-binding molecules; Petition 870250109009, dated 11 / 27 / 2025, pp. 122 / 249 114 / 190 b) a monoclonal antibody that binds to nucleic acid-binding molecules; and c) a monoclonal antibody that binds to a nucleic acid-binding protein.

[00194] The kit for detecting nucleic acid end(s) in a biological material may comprise: a) proteinase (e.g., proteinase K); b) nucleic acid-binding molecules; c) a linker molecule that binds to nucleic acid-binding molecules; d) a binding molecule that binds to a nucleic acid-binding protein; and e) additional bonding molecules.

[00195] The kit for detecting nucleic acid end(s) in a biological material may comprise: a) a hydrophilic solid support; b) nucleic acid-binding molecules; c) a linker molecule that binds to nucleic acid-binding molecules; and d) a binding molecule that binds to a nucleic acid-binding protein.

[00196] The kit for detecting nucleic acid end(s) in a biological material may comprise: a) nucleic acid-binding molecules; b) a linking molecule that binds to molecules Petition 870250109009, dated 11 / 27 / 2025, pp. 123 / 249 115 / 190 nucleic acid binding; c) a binding molecule that binds to a nucleic acid-binding protein; d) reagents to perform the amplification.

[00197] The kit for detecting nucleic acid end(s) in a biological material may comprise: a) nucleic acid-binding molecules; b) a linker molecule that binds to nucleic acid-binding molecules; c) a binding molecule that binds to a nucleic acid-binding protein; and d) additional oligonucleotide molecules.

[00198] Preferably, the hydrophilic solid support is a positively charged solid support. For example, the hydrophilic solid support could be a TOMO® slide or a slide coated with poly-L-lysine.

[00199] Nucleic acid binding molecules may comprise halogenated nucleotide or nucleoside molecules, DNA precursor analogs, and / or biotinylated nucleotide molecules. The kit may comprise at least one type of nucleic acid binding molecule. The kit may comprise at least two, at least three, or at least four types of nucleic acid binding molecules.

[00200] The bonding molecules that bind to Petition 870250109009, dated 11 / 27 / 2025, pp. 124 / 249 116 / 190 nucleic acid-binding molecules can be antibodies or fragments thereof, streptavidin molecules, avidin molecules, streptavidin analogs, biotin molecules, peptides, proteins, nucleic acids, azides, or polymers.

[00201] The two different bonding molecules can be of the same type or of different types. For example, the two different bonding molecules can be: (i) different antibodies or fragments thereof, such as a monoclonal antibody (or a fragment thereof) and a polyclonal antibody (or a fragment thereof); (ii) different antibodies or fragments thereof, such as a monoclonal antibody (or a fragment thereof) specific for a first nucleic acid-binding molecule used in the method and a monoclonal antibody (or a fragment thereof) specific for a second nucleic acid-binding molecule used in the method; (iii) an antibody or fragment thereof and streptavidin; (iv) an antibody or fragment thereof and an avidin; (v) an antibody or fragment thereof and a protein or peptide; or (vi) an antibody or fragment thereof and a biotin molecule.

[00202] Preferably, the two molecules of Petition 870250109009, dated 11 / 27 / 2025, pp. 125 / 249 117 / 190 different binding molecules are (or comprise) two different monoclonal antibodies (or fragments thereof); one specific for the nucleic acid-binding molecule used in the method and the other specific for the nucleic acid-binding protein (i.e., the intrinsic nucleic acid-binding protein). Preferably, the two different binding molecules are (or comprise) two different monoclonal antibodies (or fragments thereof) from different species (e.g., a rabbit and a mouse).

[00203] In one embodiment, the two different linker molecules may be linked to one oligonucleotide molecule. Preferably, each of the two different linker molecules is linked to an oligonucleotide molecule. The oligonucleotide molecules linked to each of the two different linker molecules are preferably different (i.e., have a different sequence).

[00204] In another embodiment, the kit may additionally comprise additional binding molecules that are linked to an oligonucleotide molecule. Additional binding molecules may be of the same type or of different types. Preferably, the additional binding molecules are of different types. For example, the additional binding molecules may be two (different) antibodies. Petition 870250109009, dated 11 / 27 / 2025, pp. 126 / 249 118 / 190 (or fragments thereof). Additional linking molecules may be specific and / or selective for binding to the two different linking molecules.

[00205] The additional oligonucleotide molecules are preferably capable of hybridizing with the oligonucleotide molecules attached to the linker molecules or to the additional linker molecules present in the kit. The additional oligonucleotide molecules may comprise the same type or different types of oligonucleotides (e.g., have the same or different sequences). Preferably, the additional oligonucleotide molecules comprise at least two types of oligonucleotides (i.e., at least two oligonucleotide sequences). The additional linker molecules may be linked to form a circular template.

[00206] The reagents for performing the amplification may comprise a polymerase (e.g., a phi29 polymerase), a dNTP mixture, a primer (e.g., a hexamer primer) and / or nuclease-free water.

[00207] The kit may additionally include reagents for fixing biological material (e.g., cells). For example, the reagent for fixing biological material may be 70% (v / v) ethanol.

[00208] The kit may additionally include a polymerase and / or a ligase. The kit may include DNA Petition 870250109009, dated 11 / 27 / 2025, pp. 127 / 249 119 / 190 polymerase I, TdT, Klenow fragment, Phu polymerase, Taq polymerase, T4 DNA polymerase, T7 DNA polymerase, T4 polynucleotide kinase and / or RNA polymerase.

[00209] The invention also provides the kit described in this document for use in the methods described herein.

[00210] The preceding detailed description is provided by way of explanation and illustration and is not intended to limit the scope of the appended claims. Many variations on the embodiments currently preferred herein illustrated will be apparent to a person skilled in the art and remain within the scope of the appended claims and their equivalents.

[00211] The invention is further detailed in the following clauses: 1. A method for detecting nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the following steps: (a) incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) in the biological material without significantly affecting the levels of a nucleic acid-binding protein in the biological material; b) adding nucleic acid-binding molecules to biological material under conditions such that the molecules of Petition 870250109009, dated 11 / 27 / 2025, pp. 128 / 249 120 / 190 nucleic acid binding sites bind to the nucleic acid end(s) on biological material; (c) adding two different binding molecules to biological material under conditions such that one of the binding molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to the nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[00212] 2. The method for detecting nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase (e.g., proteinase K) at a concentration of 6 pg / mL to 8 pg / mL for less than 2 minutes; b) adding nucleic acid binding molecules to biological material under conditions such that the nucleic acid binding molecules bind to the end(s) of Petition 870250109009, dated 11 / 27 / 2025, pp. 129 / 249 121 / 190 nucleic acid in biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[00213] 3. A method for detecting nucleic acid end(s) in a biological material containing nucleic acids, wherein the method comprises the steps of: a) Incubate the biological material with a proteinase for less than 2 minutes; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; c) adding two different linker molecules to biological material under conditions such that one of the linker molecules binds to the acid-linking molecules. Petition 870250109009, dated 11 / 27 / 2025, pp. 130 / 249 122 / 190 nucleic acid-linked to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[00214] 4. The method of any of clauses 1 to 3, wherein the biological material is, or is derived from, an animal, a plant, a protozoan, a bacterium or a virus.

[00215] 5. The method of any of clauses 1 to 4, wherein the biological material comprises a cell or fragment thereof.

[00216] 6. The method of any of clauses 1 to 5, wherein the biological material comprises fixed cells.

[00217] 7. The method of any of clauses 1 to 6, wherein the nucleic acid comprises or is DNA and / or RNA, preferably DNA.

[00218] 8. The method of any of clauses 1 to 7, wherein the nucleic acid end(s) is / are one or more DNA end(s) or one or more RNA end(s), preferably one or more DNA end(s). Petition 870250109009, dated 11 / 27 / 2025, pp. 131 / 249 123 / 190

[00219] 9. The method of any of clauses 1 to 8, wherein the nucleic acid end(s) is / are a single-stranded nucleic acid break or a double-stranded nucleic acid break.

[00220] 10. The method of any of clauses 1 to 9, wherein the nucleic acid end(s) is / are a single-strand gap, a double-strand blunt-end break, a double-strand salient break at the 3' end, or a single-strand break.

[00221] 11. The method of any of clauses 1 to 10, wherein the proteinase is a broad-spectrum proteinase.

[00222] 12. The method of any of clauses 1 to 11, wherein the proteinase is an aspartic protease, a glutamic protease, a metalloprotease, a cysteine ​​protease, a serine protease or a threonine protease, or preferably, wherein the protease is a serine protease, such as proteinase K.

[00223] 13. The method of any of clauses 1 to 12, in which the incubation of the biological material with a proteinase is carried out for at least 5 seconds, preferably at least 10 seconds and most preferably at least 15 seconds.

[00224] 14. The method of any of clauses 1 to 13, in which the incubation of the biological material with a proteinase is carried out for between 1 second and less than 2 Petition 870250109009, dated 11 / 27 / 2025, pp. 132 / 249 124 / 190 minutes, between 15 seconds and 90 seconds, between 20 seconds and 80 seconds, between 25 seconds and 70 seconds, between 30 seconds and 60 seconds, preferably the incubation of the biological material with a proteinase is carried out for between 30 seconds and 60 seconds.

[00225] 15. The method of any of clauses 1 to 14, in which the incubation of the biological material with a proteinase is 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.

[00226] 16. The method of any of clauses 1 to 15, in which the incubation of the biological material with a proteinase is carried out for about 30 seconds or about 60 seconds.

[00227] 17. The method of any of clauses 1 to 16, in which the incubation step of the biological material with a proteinase is interrupted by the addition of a proteinase inhibitor.

[00228] 18. The method of any of clauses 1 to 17, in which the incubation of the biological material with a proteinase is carried out at a temperature between 15 °C and 30 °C, preferably between 20 °C and 25 °C.

[00229] 19. The method of any of the clauses 1 to 18, in which the proteinase is in solution, in a way Petition 870250109009, dated 11 / 27 / 2025, pp. 133 / 249 125 / 190 preferred, at a concentration between 1 pg / mL and 20 pg / mL, between 3 pg / mL and 15 pg / mL, between 5 pg / mL and 10 pg / mL, or between 6 pg / mL and 8 pg / mL, preferably around 7 pg / mL.

[00230] 20. The method of clause 19, in which the solution comprises a buffer, urea and / or SDS.

[00231] 21. The method of any of clauses 1 to 20, wherein nucleic acid binding molecules are selected from a group comprising: (i) halogenated nucleotide or nucleoside molecules, such as BrdU, IdU, CldU, (ii) DNA precursor analogs, such as EdU (5-ethinyl-2'-deoxyuridine), F-ara-EdU, 5-ethinyl-2'-deoxycytidine, (iii) biotinylated nucleotide molecules, (iv) ADP-ribose molecules, (v) protein molecules, (vi) nucleotide or nucleoside molecules labeled with markers; optionally wherein the markers are selected from a group comprising fluorescent molecules, or chemiluminescent molecules, or radioisotopes, or enzyme substrates, or biotin molecules.

[00232] 22. The method of any of clauses 1 to 21, wherein the nucleic acid-binding molecules are halogenated nucleotide or nucleoside molecules, DNA precursor analogs and / or nucleotide molecules. Petition 870250109009, dated 11 / 27 / 2025, pp. 134 / 249 126 / 190 biotinylated.

[00233] 23. The method of any of clauses 1 to 22, in which nucleic acid binding molecules attach to the nucleic acid ends in the biological material by an enzyme-catalyzed addition process, for example, using DNA polymerase I, TdT, Klenow fragment, Phu polymerase, Taq polymerase, T4 DNA polymerase, T7 DNA polymerase, T4 polynucleotide kinase, or RNA polymerase.

[00234] 24. The method of any of clauses 1 to 23, wherein the two different binding molecules may be or comprise two different monoclonal antibodies (or fragments thereof).

[00235] 25. The method of any of clauses 1 to 24, in which the two different binding molecules are incubated with the biological material for at least 30 minutes, preferably 60 minutes.

[00236] 26. The method of any of clauses 1 to 27, in which the two different binding molecules are incubated with the biological material at a temperature between 15 °C and 30 °C, preferably between 20 °C and 25 °C.

[00237] 27. The method of any of clauses 1 to 26, in which the step of adding two different binding molecules to the biological material may include adding the two different binding molecules separately or in Petition 870250109009, dated 11 / 27 / 2025, pp. 135 / 249 127 / 190 set, preferably the two different linking molecules are added separately, preferably one of the two different linking molecules is added first and the other different linking molecule is added second.

[00238] 28. The method of any of clauses 1 to 27, in which the step of adding two different binding molecules to the biological material comprises the steps of: a. add one of two different binding molecules to the biological material b. Incubate one of the two different binding molecules with the biological material for at least 30 minutes, preferably 60 minutes. c. Optionally, perform a washing step to remove any unbound molecules. d. add the other of the two different binding molecules to the biological material e. Incubate the other of the two different binding molecules with the biological material for at least 30 minutes, preferably 60 minutes. f. Optionally, perform a washing step to remove any unbound molecules.

[00239] 29. The method of any of clauses 1 to 28, wherein the nucleic acid-binding protein is a protein found in biological material. Petition 870250109009, dated 11 / 27 / 2025, pp. 136 / 249 128 / 190

[00240] 30. The method of any of clauses 1 to 29, in which the nucleic acid-binding protein is linked to the nucleic acid at the nucleic acid ends, directly or indirectly.

[00241] 31. The method of clause 30, in which the nucleic acid-binding protein is linked to the nucleic acid at the nucleic acid ends indirectly through interaction with an adapter protein.

[00242] 32. The method of any of clauses 1 to 31, wherein the nucleic acid-binding protein is a nucleic acid repair protein, preferably a nucleic acid end repair protein (e.g., break).

[00243] 33. The method of any of clauses 1 to 32, where the nucleic acid binding protein is RAD51, RPA (e.g., RPA70), PMS2, MLH1, PMS1, p53, MSH2, Ataxia telangiectasia and Rad3-related protein, ATM serine / threonine kinase, RAD52, XRCC1, Proliferative cell nuclear antigen, XPC, Ku70, Ku80, Nibrin, DDB2, Bloom syndrome protein, CHEK2, RAD51C, DNA polymerase eta, Rad50, DDB1, RBBP8, FANCB, PALB2, H2AX, RAD54-like DNA repair and recombination protein yH2AX, PrimPol, REV1, terminal deoxynucleotidyl transferase, nude DNA polymerase, Fanconi anemia, complementation group C, FANCF, ERCC8, Artemis, Ubiquitin ligase, RNF4, TP53BP1, AP Petition 870250109009, dated 11 / 27 / 2025, pp. 137 / 249 129 / 190 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.

[00244] 34. The method of any of clauses 1 to 33, in which the nucleic acid-binding protein comprises post-translational modifications.

[00245] 35. The method of any of clauses 1 to 34, wherein each of the two different linking molecules is linked to an oligonucleotide molecule.

[00246] 36. The method of any of clauses 1 to 35, wherein each of the two different linking molecules is linked to a different oligonucleotide molecule.

[00247] 37. The method of any of clauses 1 to 36, wherein the method further comprises a step of adding additional linking molecules that bind, respectively, to the two different linking molecules after the step of adding the two different linking molecules.

[00248] 38. The method of clause 37, in which additional linking molecules are two different additional linking molecules. Petition 870250109009, dated 11 / 27 / 2025, pp. 138 / 249 130 / 190

[00249] 39. The method of clause 37 or 38, in which additional linking molecules are attached to an oligonucleotide molecule.

[00250] 40. The method of any of the clauses to 39, wherein each of the two different additional linking molecules is linked to a different oligonucleotide molecule.

[00251] 41. The method of any of clauses 1 to 40, wherein the method further comprises, after the step of adding two different linking molecules (or after the step of adding the additional linking molecules when dependent on clauses 37 to 40), a step of adding additional oligonucleotide molecules that hybridize with the oligonucleotide molecules linked to the two different linking molecules (or to the additional linking molecules) to form a circular template.

[00252] 42. The method of any of clauses 1 to 41, wherein the method further comprises, after the step of adding two different linking molecules (or after the step of adding the additional linking molecules), a step of adding two additional different oligonucleotide molecules that hybridize with the oligonucleotide molecules linked to the two different linking molecules (or to the additional linking molecules) to form a circular template. Petition 870250109009, dated 11 / 27 / 2025, pp. 139 / 249 131 / 190

[00253] 43. The method of clause 41 or 42, in which the circular amplification pattern is formed by linking the additional oligonucleotide molecules (or the two different additional oligonucleotide molecules).

[00254] 44. The method of any of clauses 1 to 43, wherein the method further comprises a step of blocking one or more nonspecific binding sites of molecules after the step of adding two different binding molecules to the biological material and / or the step of adding additional binding molecules.

[00255] 45. The method of any of clauses 1 to 44, wherein the detection of nucleic acid ends comprises a step of performing nucleic acid amplification to produce an amplification product that is detected.

[00256] 46. The method of clause 45, in which nucleic acid amplification is a rolling circle amplification.

[00257] 47. The method of any of clauses 1 to 46, in which the detection of the colocalization of the nucleic acid-binding protein and the nucleic acid end comprises: e.g. a proximity bonding test; h. a branched proximity hybridization assay; Petition 870250109009, dated 11 / 27 / 2025, pp. 140 / 249 132 / 190 I. a FRET detection; or j. a reaction driven by proximity to fluorophores or dyes.

[00258] 48. Kit for detecting nucleic acid end(s) in a biological material, the kit being distinguished in that it comprises: a) nucleic acid-binding molecules; b) a linker molecule that binds to nucleic acid-binding molecules; and c) a binding molecule that binds to a nucleic acid-binding protein.

[00259] 49. The kit of clause 48, wherein the kit comprises a proteinase, preferably proteinase K.

[00260] 50. Kit for detecting nucleic acid end(s) in a biological material, the kit being distinguished in that it comprises: a) nucleic acid-binding molecules; b) a monoclonal antibody that binds to nucleic acid-binding molecules; and c) a monoclonal antibody that binds to a nucleic acid-binding protein.

[00261] 51. Method for evaluating the effectiveness of a therapeutic agent, the method distinguished by the fact that it comprises the following steps: Petition 870250109009, dated 11 / 27 / 2025, pp. 141 / 249 133 / 190 (a) perform the method of any of clauses 1 to 47 on a sample obtained from an individual before administration of the therapeutic agent, b) perform the method described in any of clauses 1 to 47 on a sample obtained from the individual after administration of the therapeutic agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the therapeutic agent is effective if the amount of nucleic acid end(s) detected is greater in step a) than in step b), where steps a) and b) can be performed in any order.

[00262] 52. The method of clause 51, in which the therapeutic agent targets a protein involved in the mismatch repair (MMR) pathway.

[00263] 53. The method of clause 51 or 52, in which the therapeutic agent targets PMS1, PMS2 or MLH1.

[00264] 54. Method for evaluating the effectiveness of a therapeutic agent, the method distinguished by the fact that it comprises the following steps: a) perform the method described in any of clauses 1 to 47 on a sample obtained from an individual prior to administration of the therapeutic agent, b) perform the method of any of the clauses Petition 870250109009, dated 11 / 27 / 2025, pp. 142 / 249 134 / 190 to 47 in a sample obtained from the individual after administration of the therapeutic agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the therapeutic agent is effective if the amount of nucleic acid end(s) detected is greater in step b) than in step a), where steps a) and b) can be performed in any order.

[00265] 55. The method of clause 54, in which the therapeutic agent targets RPA or RAD51.

[00266] 56. The method of clause 54 or 55, where the sample is a cancer biopsy.

[00267] 57. The method of any of the clauses to 56, wherein the sample comprises a cancerous cell.

[00268] 58. The method of any of the clauses to 57, wherein the therapeutic agent is a drug against cancer.

[00269] 59. The method of any of the clauses to 58, in which the therapeutic agent inhibits DNA synthesis, causes nucleic acid damage and / or inhibits DNA damage repair.

[00270] 60. A method for evaluating nucleic acid damage caused by an agent, the method comprising the steps of: Petition 870250109009, dated 11 / 27 / 2025, pp. 143 / 249 135 / 190 (a) perform the method described in any of clauses 1 to 47 on a sample obtained from an individual prior to administration of, or exposure to, an agent, (b) perform the method described in any of clauses 1 to 47 on a sample obtained from the individual after administration of, or exposure to, the agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the agent causes damage to the nucleic acid if the amount of nucleic acid end(s) detected is greater in step b) than in step a), where steps a) and b) can be performed in any order.

[00271] 61. The method for detecting single-stranded nucleic acid end(s) in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase (e.g., proteinase K) at a concentration of 6 pg / mL to 8 pg / mL for less than 60 seconds; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; Petition 870250109009, dated 11 / 27 / 2025, pp. 144 / 249 136 / 190 (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[00272] 62. The method of clause 61, in which the nucleic acid-binding protein binds to the single-stranded nucleic acid end(s).

[00273] 63. The method of clause 61 or 62, wherein the nucleic acid-binding protein is a protein involved in MMR.

[00274] 64. The method of any of the clauses to 63, wherein the nucleic acid binding protein is PMS1, PMS2 and / or MLH1.

[00275] 65. The method for detecting double-stranded nucleic acid ends in a biological material containing nucleic acids may comprise the following steps: a) Incubate the biological material with a proteinase Petition 870250109009, dated 11 / 27 / 2025, pp. 145 / 249 137 / 190 to increase the accessibility of the nucleic acid end(s) to the level achieved using proteinase (e.g., proteinase K) at a concentration of 6 pg / mL to 8 pg / mL for less than 2 minutes; b) adding nucleic acid-binding molecules to biological material under conditions such that the nucleic acid-binding molecules bind to the nucleic acid end(s) on the biological material; (c) adding two different linking molecules to biological material under conditions such that one of the linking molecules binds to nucleic acid-binding molecules attached to the nucleic acid end(s) and the other linking molecule binds to a nucleic acid-binding protein that is attached to the nucleic acid at the nucleic acid end(s); and d) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

[00276] 66. The method of clause 65, in which the nucleic acid-binding protein binds to the end(s) of the double-stranded nucleic acid.

[00277] 67. The method of clause 65 or 66, in which the nucleic acid-binding protein is an involved protein Petition 870250109009, dated 11 / 27 / 2025, pp. 146 / 249 138 / 190 in RH or in the repair of double-stranded DNA damage.

[00278] 68. The method of any of the clauses 65 to 67, wherein the nucleic acid binding protein is RAD51 and / or RAD70.

[00279] These and other aspects of the invention will now be described with reference to the accompanying Figures, in which: DESCRIPTION OF THE FIGURES

[00280] Figure 1 shows an exemplary method of the present invention with PMS2 as the nucleic acid-binding protein.

[00281] Figure 2 shows an exemplary method of the present invention with PMS2 as the nucleic acid-binding protein and additional binding molecules.

[00282] Figure 3 shows an exemplary method of the present invention with PMS2 as the nucleic acid-binding protein and direct incorporation into a single strand of DNA of the nucleic acid-binding molecules.

[00283] Figure 4 shows an exemplary method of the present invention with Rad51 as the nucleic acid-binding protein.

[00284] Figure 5A shows results for Sample 3 — NT cells, sSTRIDE MMR staining (Example 1).

[00285] Figure 5B shows results for Sample 4 - cells treated with 6TG, sSTRIDE MMR staining (Example 1). Petition 870250109009, dated 11 / 27 / 2025, pp. 147 / 249 139 / 190

[00286] Figure 5C shows results of antigen 1 recovery analysis (proteinase K 2 min) with 6TG treatment.

[00287] Figure 6A shows the results of Sample 5 - untreated U2OS WT, sSTRIDE MMR staining.

[00288] Figure 6B shows the results of Sample 6 - untreated U2OS WT cells, sSTRIDE MMR staining negative control (anti-biotin antibody only).

[00289] Figure 6C shows results for Sample 7 - untreated U2OS WT cells, sSTRIDE MMR staining negative control (PMS2 antibody only).

[00290] Figure 6D shows results for Sample 8 - untreated U2OS WT cells, sSTRIDE standard assay.

[00291] Figure 7 shows results of antigen 2 recovery analysis (proteinase K 1 min).

[00292] Figure 8 shows the comparison between the sSTRIDE MMR assay and the negative controls.

[00293] Figure 9A shows results for Sample 9 - untreated U2OS WT cells, sSTRIDE MMR standard assay.

[00294] Figure 9B shows results for Sample 10 - untreated U2OS WT cells, negative control of the sSTRIDE MMR assay (PMS2 antibody only).

[00295] Figure 9C shows results for Sample 11 - untreated U2OS WT cells, positive control of the sSTRIDE MMR assay (standard procedure). Petition 870250109009, dated 11 / 27 / 2025, pp. 148 / 249 140 / 190

[00296] Figure 9D shows results of antigen 3 recovery analysis (without proteinase K).

[00297] Figure 10A shows a comparison of three antigen retrieval methods (untreated U2OS WT cells). Representative images showing the result of labeling performed with the sSTRIDE-MMR assay under different antigen retrieval conditions.

[00298] Figure 10B shows results of the antigen recovery dependence analysis.

[00299] Figure 11 shows a comparison of the intensity and frequency of foci. The upper images show the sSTRIDE MMR foci, the lower images show the foci from the sSTRIDE assay.

[00300] Figure 12A shows the intensity and frequency of foci - (U2OS WT cells, 48 ​​h treatment, antigen 2 recovery).

[00301] Figure 12B shows a comparison of Abcam antigen-antibody retrieval methods. Samples 2730 - U2OS WT cells treated for 48 h with 6TG at different concentrations, positive control of the sSTRIDE MMR assay (antigen retrieval method v2).

[00302] Figure 12A shows the comparison of antigen retrieval methods - results of quantitative analysis - comparison of antigen retrieval methods. Petition 870250109009, dated 11 / 27 / 2025, pp. 149 / 249 141 / 190

[00303] Figure 13B shows results of the ratio analysis between sSTRIDE MMR and the normal sSTRIDE procedure.

[00304] Figure 13C shows results for Sample 29 - untreated U2OS WT cells - v5 antigen recovery method.

[00305] Figure 14A shows Abcam antibody negative controls. Samples 36-39 - U2OS WT cells not treated for 48 h, negative controls of the sSTRIDE MMR assay (v2 antigen recovery method). In each enzymatic reaction the labeled agent was omitted.

[00306] Figure 14B shows results of quantitative analysis - negative controls (antigen recovery 2). Results of quantitative analysis - comparison of antigen recovery methods.

[00307] Figure 14C shows results of the ratio analysis between the negative controls and sSTRIDE MMR. The following were omitted from the control sample: - N1 - PLA, N2 - nucleotides (nts), N3 - pol I reaction, N4 - antibodies (abs), N5 - anti-PMS2, N6 - anti-biotin.

[00308] Figure 15A shows results for Samples 40-43 - untreated U2OS WT cells labeled with sSTRIDE-MMR (40-42) and sSTRIDE (43), different antigen retrieval methods were tested.

[00309] Figure 15B shows results of the quantitative analysis - antigen recovery. Results of Petition 870250109009, dated 11 / 27 / 2025, pp. 150 / 249 142 / 190 analysis - ratio between the normal sSTRIDE procedure and sSTRIDE MMR.

[00310] Figure 15C shows results for different antigen recoveries.

[00311] Figure 16A shows negative controls impact of endogenous biotin blockade time on the reading level. Results of quantitative analysis comparing controls with different biotin blockade times - in red frame, blockade time of 30 min; without frame, blockade time of 15 min.

[00312] Figure 16B shows results for Samples 46 and 52 - U2OS WT cells, negative controls of the sSTRIDE MMR assay. Difference between the reading level as a function of the blocking time.

[00313] Figure 17A shows the comparison - antigen retrieval methods. v2 - 60 s, v4 - 15 s, v5 - 30 s, v6 - 90 s.

[00314] Figure 17B shows the comparison between the sSTRIDE MMR / sSTRIDE ratios.

[00315] Figure 18A shows the comparison of the ratio of negative sSTRIDE MMR controls / sSTRIDE (Abcam antibody). The following were omitted from the control: - N1 - PLA, N2 - nucleotides, N3 - pol I reaction, N4 - antibodies, N5 - anti-PMS2, N6 - anti-biotin.

[00316] Figure 18B shows the comparison of the ratio of Petition 870250109009, dated 11 / 27 / 2025, pp. 151 / 249 143 / 190 negative controls sSTRIDE MMR / sSTRIDE (Santa Cruz Biotech antibody).

[00317] Figure 19 shows the parental HAP1 cell line versus PMS2 KO (IF assessment of staining, Abcam antibody). Representative images show an overlap of DAPI and PMS2 signals.

[00318] Figure 20 shows parental HAP1 versus PMS2 KO with sSTRIDE-MMR labeling.

[00319] Figure 21 shows parental HAP1: untreated control, positive control and negative technical controls (*treatment with 6TG 1 pM for 48 h).

[00320] Figure 22 shows HAP1 PMS2-KO: untreated control, positive control and negative technical controls (*treatment with 6TG 1 pM for 48 h).

[00321] Figure 23 shows HAP1 PMS2-KO: untreated control, positive control and negative technical controls compared with different treatments.

[00322] Figure 24 shows parental HAP1: control with increased 6TG concentration.

[00323] Figure 25 shows dSTRIDE images with and without etoposide.

[00324] Figure 26 shows results of the analysis of dSTRIDE signal data, measurement: level of DNA double-strand breaks after treatment with etoposide.

[00325] Figure 27 shows results of the analysis of Petition 870250109009, dated 11 / 27 / 2025, pp. 152 / 249 144 / 190 dSTRIDE signal data, measurement: level of DNA double-strand breaks after etoposide treatment. *In the vehicle condition, the 5% most damaged cells have >192 foci.

[00326] Figure 28 shows results of the analysis of dSTRIDE signal data, measurement: level of DNA double-strand breaks after treatment with etoposide.

[00327] Figure 29 shows results of the dSTRIDE signal data analysis, measurement: level of DNA double-strand breaks after treatment with etoposide. *in the vehicle condition, 5% of the most damaged cells have >205 foci.

[00328] Figure 30 shows results of the analysis of dSTRIDE signal data, comparison between cell lines.

[00329] Figure 31 shows Rad51-dSTRIDE staining in RH-deficient and RH-proficient cell lines.

[00330] Figure 32 shows Rad51-dSTRIDE staining in RH-proficient cell lines with and without etoposide.

[00331] Figure 33 shows results of the analysis of dSTRIDE HR signal data, measurement: level of Rad51-associated DNA double-strand breaks after treatment with etoposide.

[00332] Figure 34 shows results of the analysis of dSTRIDE HR signal data, measurement: level of double-strand breaks in DNA associated with Rad51 after treatment with Petition 870250109009, dated 11 / 27 / 2025, pp. 153 / 249 145 / 190 etoposide.

[00333] Figure 35 shows results of the analysis of dSTRIDE HR signal data, measurement: level of Rad51-associated DNA double-strand breaks after treatment with etoposide.

[00334] Figure 36 shows results of the analysis of dSTRIDE HR signal data, measurement: level of Rad51-associated DNA double-strand breaks after treatment with etoposide.

[00335] Figure 37 shows results of the analysis of dSTRIDE HR signal data, comparison between cell lines.

[00336] Figure 38 shows dSTRIDE-RPA staining in RH-proficient cell lines with and without etoposide.

[00337] Figure 39 shows results of the analysis of dSTRIDE RPA signal data, measurement: level of DNA double-strand breaks associated with RPA70 after treatment with etoposide.

[00338] Figure 40 shows results of the analysis of dSTRIDE RPA signal data, measurement: level of DNA double-strand breaks associated with RPA70 after treatment with etoposide.

[00339] Figure 41 shows results of the data analysis of dSTRIDE RPA signals, measurement: level of DNA double-strand breaks associated with RPA70 after treatment with Petition 870250109009, dated 11 / 27 / 2025, pp. 154 / 249 146 / 190 etoposide.

[00340] Figure 42 shows results of the analysis of dSTRIDE RPA signal data, measurement: level of DNA double-strand breaks associated with RPA70 after treatment with etoposide.

[00341] Figure 43 shows results of the analysis of dSTRIDE RPA signal data, comparison between cell lines.

[00342] Figure 44 shows the comparison between dSTRIDE, dSTRIDE-HR (Rad51) and dSTRIDE-RPA in an RH-proficient cell line treated with etoposide.

[00343] Figure 45 shows the comparison between dSTRIDE, dSTRIDE-HR (Rad51) and dSTRIDE-RPA in an RH-deficient cell line treated with etoposide.

[00344] Figure 46 shows results of quantitative analysis in HAP1 cells (WT and KO) for sSTRIDE-MLH1.

[00345] Figure 47 shows results of quantitative analysis in HAP1 cells for sSTRIDE-MLH1 with or without 6TG treatment.

[00346] Figure 48 shows the comparison between the results of PMS2 and MLH1.

[00347] Figure 49 shows results of quantitative analysis in HAP1 cells (WT and KO) for sSTRIDE-PMS1.

[00348] Figure 50 shows the comparison between the results of PMS2, PMS1 and MLH1. Petition 870250109009, dated 11 / 27 / 2025, pp. 155 / 249 147 / 190 EXAMPLES

[00349] The present invention is explained in more detail with the aid of the following examples, which are not intended to limit the scope of the present invention in any way.

[00350] The examples are described based on general protocols that relate to the main modalities presented in the description. Example 1 - PMS1, PMS2 or MLH1 detection protocol Sample pretreatment - optional (optional) fixation of biological material in 70% (v / v) ethanol Proteinase treatment 1. Treatment of biological material with proteinase K for 30 seconds (at a concentration of approximately 7 pg / mL (1:350 dilution of the 2.5 mg / mL proteinase K stock concentration), solution in PBS) at approximately 22 °C (i.e., room temperature) 2. (Optional) Incubation with 1 mM PMSF in PBS for 1 minute at approximately 22°C. 3. (Optional) Rinse with PBS. DNA end modification - optional 1. (Optional) Reaction with T4 PNK Endogenous biotin blocking step - optional 1. (optional) 30-minute incubation in solution of Petition 870250109009, dated 11 / 27 / 2025, pp. 156 / 249 148 / 190 streptavidin blockade at approximately 22°C 2. (Optional) Rinse with PBS. 3. (Optional) 30-minute incubation in biotin blocking solution at approximately 22°C. 4. (Optional) Rinse with PBS. Modified nucleotide linkage with biotin 5. Polymerase I reaction at 37 °C with polymerase I enzyme, biotin-7-ATP, biotin-16-dUTP, biotin-16dCTP and dGTP. 6. (Optional) Rinse with a tampon. Antibody binding 1. (Optional) 60-minute incubation in Navinci blocking buffer at 37°C. 2. Incubate for 60 minutes in rabbit monoclonal anti-PMS2 (or anti-PMS1 or anti-MLH1) antibody at a 1:100 dilution in Navinci primary antibody diluent at approximately 22°C. 3. (Optional) Rinse with PBS. 4. Incubation for 60 minutes in mouse monoclonal anti-biotin antibody at a 1:100 dilution in Navinci primary antibody diluent at approximately 22°C. 5. (Optional) Rinse with PBS. PLA Procedure 1. Binding reaction of PLA probes at 37 °C (Navinci PLA reagents and standard protocol) Petition 870250109009, dated 11 / 27 / 2025, pp. 157 / 249 149 / 190 2. Reaction A at 37°C 3. Reaction B at 37°C 4. Reaction C at 37°C List of reagents - as used in Examples 1 Reagent Step Manufacturer Serial Number Proteinase Proteinase K Novus Biological NB900-66727 PMSF Sigma-Aldrich P7626 T4 PNK Buffer T4 PNK New England BioLabs B0201S Enzyme T4 PNK New England BioLabs M0201 Blocking Streptavidin Invitrogen 21390 Biotin Invitrogen E21390 Reaction Pol I Pol I (3U) New England BioLabs M0209L Buffer (NEB2) New England BioLabs B7002S Biotin-7-dATP Jena Bioscience NU-835-BIO-L Biotin-16-dUTP Jena Bioscience NU-803-BIO-L Biotin-16-dCTP Jena Bioscience NU-809-BIO-16-L dGTP Jena Bioscience NU-1003S Blocking Buffer Navinci Navinci NaveniFlex MR PLA Probe Diluent Probe M1 Probe R2 Enzyme A Buffer A Enzyme B Buffer B Enzyme C Buffer C Table 1. Details of the reagents Antibody Manufacturer Serial Number Anti-biotin antibody [Hyb-8] Ms mAb for biotin Abcam ab201341 Rb pAb for biotin Abcam ab53494 Anti-BrdU antibody [MoBu-1] Abcam ab8039 Rb pAb for BrdU Abcam ab152095 Recombinant anti-PMS2 antibody [EPR3947] Abcam ab214442 Recombinant anti-Rad51 antibody [EPR4030(3)] Abcam ab221796 Recombinant anti-RPA70 antibody [EPR3472] Abcam ab79398 Table 2. Antibody details Example 2 - Rad51 detection protocol Petition 870250109009, dated 11 / 27 / 2025, pp. 158 / 249 150 / 190 Sample pretreatment - optional (optional) fixation of biological material in 70% (v / v) ethanol Proteinase treatment 1. Treatment of the biological material with proteinase K for 60 seconds (at a concentration of approximately 7 pg / mL (1:350 dilution of the proteinase K stock concentration of 2.5 mg / mL), solution in PBS) at approximately 22 °C (i.e., room temperature). 2. (Optional) Incubation with 1 mM PMSF in PBS for 5 minutes at approximately 22°C. 3. (Optional) Rinse with PBS. DNA end modification - optional 1. (Optional) Reaction with T4 PNK TdT reaction - BrdU incorporation 1. TdT reaction at 37°C with BrdU and TdT enzyme 2. (Optional) Rinse with PBS. Antibody binding 6. (Optional) 60-minute incubation in Navinci blocking buffer at 37°C. 7. Incubation for 60 minutes in rabbit anti-Rad51 monoclonal antibody at a 1:100 dilution in Navinci primary antibody diluent at approximately 22°C. 8. (Optional) Rinse with PBS. 9. 60-minute incubation in monoclonal antibody. Petition 870250109009, dated 11 / 27 / 2025, pp. 159 / 249 151 / 190 mouse anti-BrdU at a 1:100 dilution in Navinci primary antibody diluent at approximately 22°C 10. (Optional) Rinse with PBS. PLA Procedure 1. Binding reaction of PLA probes at 37 °C (Navinci PLA reagents and standard protocol) 2. Reaction A at 37°C 3. Reaction B at 37°C 4. Reaction C at 37°C Example 3 - RPA70 detection protein Sample pre-treatment - optional 1. (Optional) Fixation of biological material in 70% (v / v) ethanol. Proteinase treatment 1. Treatment of the biological material with proteinase K for 60 seconds (at a concentration of approximately 7 pg / mL (1:350 dilution of the proteinase K stock concentration of 2.5 mg / mL, solution in PBS) at approximately 22 °C (i.e., room temperature) 2. (Optional) Incubation with 1 mM PMSF in PBS for 1 minute at approximately 22°C. 3. (Optional) Rinse with PBS. DNA end modification - optional 1. (Optional) Reaction with T4 PNK TdT reaction - BrdU incorporation Petition 870250109009, dated 11 / 27 / 2025, pp. 160 / 249 152 / 190 1. TdT reaction at 37°C with BrdU and TdT enzyme 2. (Optional) Rinse with PBS. Antibody binding 1. (Optional) 60-minute incubation in Navinci blocking buffer at 37°C. 2. Incubate for 60 minutes in rabbit anti-RPA70 monoclonal antibody at a 1:100 dilution in Navinci primary antibody diluent at approximately 22°C. 3. (Optional) Rinse with PBS. 4. Incubation for 60 minutes in mouse anti-BrdU monoclonal antibody at a 1:100 dilution in Navinci primary antibody diluent at approximately 22°C. 5. (Optional) Rinse with PBS. PLA Procedure 1. Binding reaction of PLA probes at 37 °C (Navinci PLA reagents and standard protocol) 2. Reaction A at 37°C 3. Reaction B at 37°C 4. Reaction C at 37°C Example 4 - Optimization of protease treatment Objective of the experiments

[00351] Experimental setup and optimization: Detection and quantitative analysis of PMS2-activated DNA single-strand breaks in human U2OS WT cells. Methods Petition 870250109009, dated 11 / 27 / 2025, pp. 161 / 249 153 / 190 cells U2OS

[00352] USOS cells were grown in plates T25 Falcon (Nunc) in DMEM HG medium (Gibco) containing 10% FBS (Gibco) and 100 U / mL penicillin and 100 μg / mL streptomycin (Gibco). Antigen retrieval method - treatment with proteinase K

[00353] Proteinase K was used from a freshly opened vial. The antigen retrieval method number corresponds to the numbers in the table below. 1. v1 proteinase K - 2 min incubation, 2. v2 proteinase K - 1 min incubation, 3. v3 without proteinase, 4. v4 proteinase K - 15s incubation, 5. v5 proteinase K - 30s incubation, 6. v6 proteinase K - 90s incubation, 7. v7 proteinase K - 180s incubation. Anti-PMS2 antibody

[00354] Rabbit recombinant anti-PMS2 antibody [EPR3947] - BSA and azide free (ab214442)

[00355] Mouse anti-PMS2 monoclonal antibody (B3): sc-25315 Condition tested - five optimization experiments sample cell line composite assay treatment time (h) antigen recovery Petition 870250109009, dated 11 / 27 / 2025, pp. 162 / 249 154 / 190 EXP1 1 U2OS WT IF PMS2 vehicle 0 X 2 U2OS WT IF PMS2 6TG 48h X EXP2 3 U2OS WT As in Example 1 vehicle 0 1 4 U2OS WT As in Example 1 6TG 48h 1 EXP3 5 U2OS WT As in Example 1 vehicle 0 2 6 U2OS WT* As in Example 1 vehicle 0 2 7 U2OS WT* As in Example 1 vehicle 0 2 8 U2OS WTA As in Example 1 vehicle 0 2 EXP4 9 U2OS WT As in Example 1 vehicle 0 3 10 U2OS WT* As in Example 1 vehicle 0 3 11 U2OS WTA sSTRIDE vehicle 0 3 12 U2OS WT As in Example 1 6TG 48 3 13 U2OS WT* As in Example 1 6TG 48 3 14 U2OS WTA sSTRIDE 6TG 48 3 * negative controlsΛpositive controls

[00356] In the sSTRIDE method, all steps of the Example are performed except that the method is not based on the detection of nucleic acid-binding molecules. Petition 870250109009, dated 11 / 27 / 2025, pp. 163 / 249 155 / 190 intrinsic and instead focuses solely on the detection of the nucleic acid end(s). Thus, in the sSTRIDE method, the standard procedure is to apply proteinase treatment. K for 3 min (same concentration, same temperature as in Example 1). The (optional) endogenous biotin blocking step is performed for 15 min (each) instead of 30 min (each). Instead of anti-PMS2 antibodies and anti-biotin antibodies, two anti-biotin antibodies are used (each with different affinity and / or different binding site on the biotin molecule).

[00357] The same reagents were used in each experiment (see reagents in Example 1).

[00358] The endogenous biotin streptavidin / biotin kits were the same in each experiment.

[00359] The same batch of proteinase K was used for each sample. Abcam antibody staining procedure

[00360] The samples were processed according to standard sSTRIDE protocols (as above) with the following modifications: 1. Different antigen retrieval methods were tested. 2. One of the anti-biotin antibodies was replaced with PMS2 antibody — sSTRIDE MMR assay. 3. Positive control - tests were performed Petition 870250109009, dated 11 / 27 / 2025, pp. 164 / 249 156 / 190 sSTRIDE standard. Negative controls were performed - one of the antibodies was discarded. Incubation was carried out in a 1% BSA solution without antibody. Experiment Code: STRIDE MMR Optimization Assay Type: In vitro STRIDE sSTRIDE MMR PMS2 LB1 45 min EDTA 10mM 15 min EDTA 50mM 7 min Proteinase Conditions: The table below varies between experiments. Biotin / streptavidin 15 min mono Anti-PMS2 Ab Rb 1:100, 60 min (Abeam) poly Anti-biotin Ab Ms 1:100, 60 min (Abeam) Counterstain: DAPI 500 nM, 15 min Treatment: 6TG 10nM 48h, vehicle Additional Staining: X Table 3. Method details. Rb - rabbit (host) antibody, Ms - mouse (host) antibody. Staining procedure: antibody from Santa Cruz Biotechnology

[00361] The samples were processed according to the standard sSTRIDE protocols with the following modifications: 1. Different antigen retrieval methods were tested. Petition 870250109009, dated 11 / 27 / 2025, pp. 165 / 249 157 / 190 2. One of the anti-biotin antibodies was replaced by PMS2 antibody - sSTRIDE MMR assay. 3. Positive control - standard sSTRIDE assays were performed. 4. Negative controls were performed - one of the antibodies was discarded. Incubation was carried out in a 1% BSA solution without antibody. Experiment Code: STRIDE MMR Optimization Assay Type: In vitro STRIDE sSTRIDE MMR PMS2 LB1 45 min EDTA 10mM 15 min EDTA 50mM 7 min Proteinase Conditions: The table below varies between experiments. Biotin / Streptavidin 15 min mono Anti-PMS2 Ab Ms 1:100, 60 min (Santa Cruz Biotechnology) poly Anti-biotin Ab Rb 1:100, 60 min (Abeam) Counterstain: DAPI 500 nM, 15 min Treatment: 6TG 10nM 48h, vehicle Additional Staining: X Table 4. Method details. Rb - rabbit (host) antibody, Ms - mouse (host) antibody. Results, Part 1 EXP2 sample number treatment 1st antibody 2nd antibody antigen recovery Petition 870250109009, dated 11 / 27 / 2025, pp. 166 / 249 158 / 190 3 As in Example 1 - sSTRIDE MMR vehicle PMS2 Rb Biotin Ms 1 4 As in Example 1 - sSTRIDE MMR 6TG PMS2 Rb Biotin Ms 1 Table 5. Conditions tested

[00362] Figure 5A - Sample 3 - NT cells, sSTRIDE MMR staining (Example 1). A low number of foci was observed, with a maximum of 3 foci per nucleus (antigen recovery 1).

[00363] Figure 5B - Sample 4 - cells treated with 6TG, sSTRIDE MMR staining (Example 1). A low number of foci was observed, with a maximum of 3 foci per nucleus (antigen recovery 1).

[00364] Figure 5C - Results of antigen 1 recovery analysis (proteinase K 2 min). Quantitative image analysis shows that the number of foci detected is low and no response was observed after treatment with 6TG. EXP3 sample nr treatment 1st antibody 2nd antibody antigen recovery 5 As in Example 1 - sSTRIDE MMR vehicle PMS2 Rb Biotin Ms 2 6 As in Example 1 - vehicle BSA Biotin Ms 2 Petition 870250109009, dated 11 / 27 / 2025, pp. 167 / 249 159 / 190 sSTRIDE MMR * 7 As in Example 1 - sSTRIDE MMR * vehicle PMS2 Rb BSA 2 8 As in Example 1 - sSTRIDE MMR Λ vehicle Biotin Rb Biotin Ms 2 Table 6. Conditions tested

[00365] Figure 6A - Sample 5 - Untreated U2OS WT, sSTRIDE MMR staining. More foci are observed in antigen recovery method 2 compared to method 1. Low amount of background signal is detected (antigen recovery 2).

[00366] Figure 6B - Sample 6 - untreated U2OS WT cells, sSTRIDE MMR staining negative control (anti-biotin antibody only). Individual foci can be detected (antigen recovery 2).

[00367] Figure 6C - Sample 7 - untreated U2OS WT cells, sSTRIDE MMR staining negative control (PMS2 antibody only). No focus detected, elevated background signal visible (antigen recovery 2).

[00368] Figure 6D - Sample 8 - untreated U2OS WT cells, standard sSTRIDE assay. Numerous foci can be detected in a single nucleus — more than 20 per nucleus (antigen recovery 2).

[00369] Figure 7 - Analysis results Petition 870250109009, dated 11 / 27 / 2025, pp. 168 / 249 160 / 190 antigen recovery 2 (proteinase K 1 min). Quantitative image analysis showing comparison of readings in U2OS cells after labeling with sSTRIDE and sSTRIDE-MMR assays and negative controls. Results show that sSTRIDE-MMR foci constitute approximately 12% of total SSBs, while signals in negative controls remain at low levels.

[00370] Figure 8 - Comparison between the sSTRIDE MMR assay and negative controls. Samples were stained and imaged on the same day. Comparison of intensity and frequency of foci (U2OS WT cells, NT, antigen 2 recovery). Images show that very low numbers of foci were detected in the negative controls when one of the antibodies (directed to biotin or PMS2) was absent. EXP4 sample nr treatment 1st antibody 2nd antibody antigen recovery 9 As in Example 1 - sSTRIDE MMR Vehicle PMS2 Rb Biotin Ms 3 10 sSTRIDE MMR Vehicle PMS2 Rb BSA 3 11 sSTRIDE Vehicle Biotin Rb Biotin Ms 3 12 sSTRIDE MMR 6TG PMS2 Rb Biotin Ms 3 13 sSTRIDE MMR 6TG PMS2 Rb BSA 3 14 sSTRIDE 6TG Biotin Rb Biotin Ms 3 Table 7. Conditions tested

[00371] Figure 9A - Sample 9 - U2OS WT cells not Petition 870250109009, dated 11 / 27 / 2025, pp. 169 / 249 161 / 190 treated, standard sSTRIDE MMR assay. Individual foci can be detected in the nucleus, high background intensity is observed (antigen recovery 3).

[00372] Figure 9B - Sample 10 - untreated U2OS WT cells, negative control of the sSTRIDE MMR assay (PMS2 antibody only). No signal can be detected (antigen recovery 3).

[00373] Figure 9C - Sample 11 - untreated U2OS WT cells, positive control of the sSTRIDE MMR assay (standard procedure). More than 10 signals per nucleus can be detected (antigen recovery 3).

[00374] Figure 9D - Results of antigen 3 recovery analysis (without proteinase K). The results of quantitative image analysis show that, in the absence of proteinase treatment, the number of foci detected in sSTRIDE-MMR (PMS2) and sSTRIDE is low. Summary - Part 1

[00375] In the studies, the best antigen recovery method for sSTRIDE-MMR-PMS2 proved to be method 2 (1 minute incubation with proteinase K). In the cases of method 1 and method 3, fewer foci were detected.

[00376] Figure 10A - Comparison of three antigen retrieval methods (untreated U2OS WT cells). Representative images showing the result of labeling performed with the sSTRIDE-MMR assay under different conditions. Petition 870250109009, dated 11 / 27 / 2025, pp. 170 / 249 162 / 190 antigen retrieval. Different numbers of fluorescence foci are visible for various antigen retrieval conditions, with the highest number of foci for v2 antigen retrieval.

[00377] Figure 10B - Results of antigen recovery dependence analysis. Quantitative image analysis confirms that the highest number of foci is detected when method 2 is applied.

[00378] The sSTRIDE assay without proteinase (antigen retrieval 3) is less effective than the assay with proteinase condition. The number of foci per nucleus is 90% lower than after 1 minute of treatment with proteinase (antigen retrieval 3).

[00379] The background level from the PMS2 antibody is negligible in the assay under each condition.

[00380] The background level from the biotin antibody cannot be considered negligible in the assay. The average foci count per nucleus is 0.9; however, in some nuclei the count level is greater than 5.

[00381] Differences between the number and morphology of the foci were detected in the tests.

[00382] Figure 11 - Comparison of intensity and frequency of foci. The upper photos show the foci from the sSTRIDE-MMR test, and the lower photos show the foci from the Petition 870250109009, dated 11 / 27 / 2025, pp. 171 / 249 163 / 190 sSTRIDE assay. sSTRIDE-MMR foci are more intense, but less frequent (U2OS WT cells, NT, antigen recovery 1). Conclusions - Part 1 1. The antigen retrieval method has a significant impact on both the standard sSTRIDE assay and the sSTRIDEMMR2 assay. 2. The antigen retrieval step is relevant in the sSTRIDE-MMR-PMS2 assay — without proteinase the assay does not function. 3. The best tested method of antigen retrieval is the proteinase K condition for 1 minute. 4. The background level from the biotin antibody is not negligible — the negative control with biotin antibody is relevant in each sSTRIDE-MMRPMS2 staining. Results, Part 2 EXP5 Abcam Ab nr sample treatment 1st antibody 2nd antibody antigen recovery 15 As in Example 1 - sSTRIDE MMR 6TG, 250 nM, 24h PMS2 biotin v2 16 As in Example 1 - sSTRIDE MMR 6TG, 500 nM, 24h PMS2 biotin v2 17 As in Example 1 - sSTRIDE MMR 6TG, 750 nM, 24h PMS2 biotin v2 18 As in Example 1 6TG, 1 pM, 24h PMS2 biotin v2 Petition 870250109009, dated 11 / 27 / 2025, pp. 172 / 249 164 / 190 - sSTRIDE MMR 19 As in Example 1 - sSTRIDE MMR 6TG, 5 pM, 24h PMS2 biotin v2 20 As in Example 1 - sSTRIDE MMR 6TG, 10 pM, 24h PMS2 biotin v2 21 As in Example 1 - sSTRIDE MMR 6TG, 250 nM, 48h PMS2 biotin v2 22 As in Example 1 - sSTRIDE MMR 6TG, 500 nM, 48h PMS2 biotin v2 23 As in Example 1 - sSTRIDE MMR 6TG, 750 nM, 48h PMS2 biotin v2 24 As in Example 1 - sSTRIDE MMR 6TG, 1 pM, 48h PMS2 biotin v2 25 As in Example 1 - sSTRIDE MMR 6TG, 5 pM, 48h PMS2 biotin v2 26 As in Example 1 - sSTRIDE MMR 6TG, 10 pM, 48h PMS2 biotin v2 27 As in Example 1 - sSTRIDE MMR vehicle PMS2 biotin v4 28 As in Example 1 - sSTRIDE MMR vehicle PMS2 biotin v5 29 As in Example 1 - sSTRIDE MMR vehicle PMS2 biotin v2 30 As in Example 1 - sSTRIDE MMR vehicle PMS2 biotin v6 31 sSTRIDE vehicle biotin biotin v2 32 sSTRIDE vehicle biotin biotin v7 33 As in Example 1 - sSTRIDE MMR vehicle without PLA v2 Petition 870250109009, dated 11 / 27 / 2025, pp. 173 / 249 165 / 190 34 As in Example 1 - sSTRIDE MMR vehicle without polymerase I v2 35 As in Example 1 - sSTRIDE MMR vehicle without nucleotides v2 36 As in Example 1 - sSTRIDE MMR vehicle without polymerase I reaction v2 37 As in Example 1 - sSTRIDE MMR vehicle without antibodies v2 Table 8. Conditions tested

[00383] Samples 15-18 and 21 were not visualized in 3D because the maximum number of foci observed per nucleus was 2 to 3, therefore, this condition cannot be considered the positive control of the assay. Conclusions - Part 2

[00384] Cells treated with 6TG did not respond as expected. The level of readings is lower than in untreated samples. In each condition, antigen retrieval method 2 was used. However, in some cells, the foci were very bright and large, which may lead to the conclusion that MMR is more intense in specific locations and that the detected foci are multiple.

[00385] Figure 12A - Intensity and frequency of foci — (U2OS WT cells, 48 ​​h treatment, antigen 2 recovery). The morphology of the foci is as expected, with a high signal-to-noise ratio. Petition 870250109009, dated 11 / 27 / 2025, pp. 174 / 249 166 / 190

[00386] Figure 12B - Comparison of Abcam antigen-antibody retrieval methods. Samples 27-30 U2OS WT cells treated for 48 h with 6TG at different concentrations, positive control of the sSTRIDE MMR assay (antigen retrieval method v2). Treatment did not significantly increase the number of foci in the nucleus.

[00387] Figure 13A - Comparison of antigen retrieval methods. Results of quantitative analysis - comparison of antigen retrieval methods.

[00388] Figure 13B - Analysis results - ratio between sSTRIDE MMR and the normal sSTRIDE procedure. The most promising antigen retrieval method is v5 - 30 seconds. In this sample, the signal distribution is different - some groups of nuclei show more than 40 foci per nucleus.

[00389] Figure 13C - Sample 29 - untreated U2OS WT cells - v5 antigen retrieval method. v5 antigen retrieval was chosen as the best antigen retrieval method.

[00390] Figure 14A - Negative controls - Abcam antibody. Samples 36-39 - U2OS WT cells untreated for 48 h, negative controls of the sSTRIDE MMR assay (v2 antigen recovery method). In each enzymatic reaction, the labeled agent was omitted.

[00391] Figure 14B - Analysis results Petition 870250109009, dated 11 / 27 / 2025, pp. 175 / 249 167 / 190 quantitative - negative controls (antigen recovery 2). Results of quantitative analysis - comparison of antigen recovery methods.

[00392] In the graph, the result of the counts for sample 37 is not shown. The number of SSB per nucleus is 3.8. The number of foci in most controls can be considered negligible. However, the number of SSB in the negative controls with polymerase I may lead to the conclusion that, in the assay, the blocking time of endogenous biotin should be increased.

[00393] The ratio between the sSTRIDE-MMR assay and the procedure also shows a significant amount of signals in the negative control with the polymerase I enzyme omitted (not shown in the graph - ratio equal to 0.49).

[00394] Figure 17C - Analysis results - ratio between negative controls and sSTRIDE MMR. The following were omitted from the control sample: - N1 - PLA, N2 - nucleotides (nts), N3 - poly reaction, N4 - antibodies (abs), N5 - anti-PMS2, N6 - anti-biotin. Results, part 3 EXP6 antibody from Santa Cruz Biotechnology Sample Nr Treatment 1st antibody 2nd antibody Antigen recovery 38 As in Example 1 - sSTRIDE MMR 6TG, 1 μM, 48h PMS2 Ms Biotin Rb v2 39 As in Example 6TG, 10 μM, 48h PMS2 Ms Biotin Rb v2 Petition 870250109009, dated 11 / 27 / 2025, pp. 176 / 249 168 / 190 1 - sSTRIDE MMR 40 As in Example 1 - sSTRIDE MMR vehicle PMS2 Ms Biotin Rb v5 41 As in Example 1 - sSTRIDE MMR vehicle PMS2 Ms Biotin Rb v2 42 As in Example 1 - sSTRIDE MMR vehicle PMS2 Ms Biotin Rb v6 43 sSTRIDE vehicle Biotin Ms Biotin Rb v2 44 sSTRIDE vehicle Biotin Ms Biotin Rb v7 45 As in Example 1 - sSTRIDE MMR vehicle without PLA v2 46 As in Example 1 - sSTRIDE MMR vehicle without polymerase I enzyme v2 47 sSTRIDE As in Example 1 - sSTRIDE MMR vehicle without nucleotides v2 48 As in Example 1 - sSTRIDE MMR vehicle without polymerase I reaction v2 49 As in Example 1 - sSTRIDE MMR vehicle without antibodies v2 50 As in Example 1 - sSTRIDE MMR vehicle x Biotin Rb v2 51 As in Example 1 - sSTRIDE MMR vehicle PMS2 Ms x v2 Petition 870250109009, dated 11 / 27 / 2025, pp. 177 / 249 169 / 190 52 As in Example 1 - sSTRIDE MMR vehicle without nucleotides v2 53 As in Example 1 - sSTRIDE MMR vehicle without polymerase I reaction v2 Table 9. Conditions tested

[00395] Sample 39 was not obtained in 3D because the maximum number of foci observed per nucleus was 2 to 3, therefore, this condition cannot be the positive control of the assay.

[00396] Samples 52 and 53 were stained with the Abcam antibody. In the procedure, the endogenous biotin blocking time was changed to 30 minutes (15 minutes longer than in previous experiments).

[00397] Figure 15A - Samples 40-43 - U2OS cells Untreated WT labeled with sSTRIDE-MMR (40-42) and sSTRIDE (43), different antigen retrieval methods were tested.

[00398] Figure 15B - Results of quantitative analysis - antigen recovery. Results of the analysis - ratio between the normal sSTRIDE procedure and sSTRIDE MMR.

[00399] Figure 15C - In experiment 05, the most promising versions of the antigen retrieval procedure Petition 870250109009, dated 11 / 27 / 2025, pp. 178 / 249 170 / 190 were tested. The v5 antigen recovery shows the highest amount of signal per nucleus (the same version was chosen in the experiments with the Abcam antibody).

[00400] Figure 16A - Negative controls - impact of endogenous biotin blockade time on reading level. Results of quantitative analysis - comparison of controls with different biotin blockade times - in red frame, blockade time of 30 min; without frame, blockade time of 15 min.

[00401] Figure 16B - Results for Samples 46 and 52 - U2OS WT cells, negative controls of the sSTRIDE MMR assay. Difference between the reading level as a function of the blocking time.

[00402] Figure 17A - Comparison - antigen retrieval methods. v2 - 60 s, v4 - 15 s, v5 - 30 s, v6 - 90 s. The highest reading is observed for 30 seconds of treatment with proteinase K. Higher readings are observed for the antibody from Santa Cruz Biotechnology.

[00403] Figure 17B - Comparison between sSTRIDE MMR / sSTRIDE ratios. The ratio is highest for both antibodies in v5 antigen recovery - 30 seconds of proteinase K treatment.

[00404] Figure 18A - Comparison of the ratio of negative sSTRIDE MMR controls / sSTRIDE (Abcam antibody). The following were omitted in the control: - N1 - PLA, N2 - nucleotides, N3 - reaction Petition 870250109009, dated 11 / 27 / 2025, pp. 179 / 249 171 / 190 poll, N4 - antibodies, N5 - anti-PMS2, N6 - anti-biotin. The ratio for all negative controls is at a very low level, confirming the specificity of the assay.

[00405] Figure 18B - Comparison of the ratio of negative controls sSTRIDE MMR / sSTRIDE (Santa Cruz Biotech antibody). The ratio is higher than in the case of the Abcam antibody, which shows that the Santa Cruz Biotechnology antibody is characterized by more nonspecific binding. Conclusions - Part 3

[00406] Both Abcam and Santa Cruz Biotechnology antibodies can be used in the sSTRIDEMMR assay. The highest readings are obtained with the Santa Cruz Biotechnology antibodies; however, these antibodies also result in higher readings in the negative controls. Increasing the endogenous biotin blockade time may help reduce the background signal observed in the negative controls. Example 5 - Development of the sSTRIDE-MMR assay variant (PMS2) A. Cell culture conditions - HAP1 cells

[00407] The HAP1 cell line is a near-haploid human cell line derived from chronic myeloid leukemia (CML). The advantages of the HAP1 cell line in assay development research are: one copy of each specific allele, fast doubling time, and ease of use. Petition 870250109009, dated 11 / 27 / 2025, pp. 180 / 249 172 / 190 transfection. The parental HAP1 and HAP1 knockout strains for PMS2 are offered as an isogenic pair. The PMS2 knockout protein was achieved by CRISPR / Cas editing to contain a 1 bp insertion in a PMS2 coding exon.

[00408] HAP1 and HAP1 PMS2 KO cells were acquired from the Horizon Discovery cell culture collection (May 18, 2022). A detailed description of the cell culture is listed in Table 10. The cells were cultured at 37 °C with 5% CO2 in T75 flasks with IMDM + 10% FBS + 1% penicillin-streptomycin solution (10,000 U each). The master bank was prepared from passage #1 performed in the intoDNA laboratory.

[00409] The cryopreservation procedure was performed as described below. The cell suspension was centrifuged at room temperature (1000 rpm, 5 min). Then, the medium was removed and a sterile mixture containing 60% medium, 30% FBS, and 10% DMSO was added. The mixture was gently homogenized and pipetted into cryotubes (each cryotube contains 800,000 cells). The cryotube was transferred to -80 °C in a Nalgene Mr. Frosty slow-freeze container (temperature reduction of approximately 1 °C per minute). The experiments included in this report were performed using the cell bank prepared on June 6, 2022. Petition 870250109009, dated 11 / 27 / 2025, pp. 181 / 249 173 / 190 Cell line Origin of the cell line Manufacturing batch number Parental HAP1 batch number Human near-haploid cell line 51523 29281 HAP1 PMS2 KO Human near-haploid cell line 43674 29280 Table 10. A list of cell phone banks acquired from ATCC. B. Seeding and treatment protocol Seeding protocol

[00410] When cell confluence reached 90%, the cells were washed with sterile PBS (without Ca2+ and Mg2+) and incubated for 2 minutes with 2 mL of 0.25% trypsin solution with EDTA. The trypsin was inactivated with the culture medium containing FBS, and the collected cells were counted in a Burker chamber. The cells were then seeded onto coverslips in 12-well plates at the densities listed in Table 11 (including the number of passages). The cells were then left to stand for 24 hours in the incubator. Name HD Modification Passage Number Seeding Density (number of cells / well) HAP1 Parental #4-#6 30k HAP1 PMS2 KO #3-#5 50k Table 11. A list of cell seeding densities. The same densities were used for the sSTRIDE and sSTRIDE MMR samples. The number of passages corresponds to the passage performed in the intoDNA laboratory. Petition 870250109009, dated 11 / 27 / 2025, pp. 182 / 249 174 / 190 Treatment protocol

[00411] After 24 hours of rest, the operating medium was replaced with fresh medium supplemented with 1 μM or 10 μM of 6-Thioguanine (6TG) in DMSO and incubated for 48 hours (the final DMSO concentration in the medium was 0.1% in each experimental condition). Under vehicle conditions, the operating medium was replaced with fresh medium supplemented with 0.1% sterile DMSO and left to stand for the same incubation time. Then, all samples were fixed with ice-cold 70% ethanol and stored at -20 °C.

[00412] *The 6TG stock was freshly prepared from powder. The powder was suspended in sterile DMSO to a final stock concentration of 100 mM. The 6TG solution in DMSO was stored in aliquots at -20 °C in the dark. C. Reagents Reagent Manufacturer Serial Number Fetal bovine serum Gibco 16140071 IMDM Gibco 1244053 Penicillin-streptomycin solution (10,000 units each) Gibco 15140130 Trypsin-EDTA (0.25%), phenol red Gibco 25200056 PBS, pH 7.2 Gibco 20012027 Dimethyl sulfoxide bioreagent Thermo Scientific J66650.AD DAPI solution Thermo Scientific 62248 6-Thioguanine (6TG) Sigma Aldrich A4882-1G Table 12. List of reagents used in procedures Petition 870250109009, dated 11 / 27 / 2025, pp. 183 / 249 175 / 190 cell culture. D. List of antibodies Antibody Manufacturer Serial Number Dilution Anti-biotin antibody [Hyb-8] Ms mAb for biotin Abcam ab201341 1:100 Rabbit pAb for Biotin Abcam ab53494 1:100 Recombinant anti-PMS2 antibody [EPR3947] Abcam ab214442 1:100 Mouse PMS2 antibody (B-3) SCB sc-25315 1:100 Recombinant anti-MLH1 antibody [EPR3894] Abcam ab92312 1:100 PMS1 antibody (PCRP-PMS1-2E11) Novus NBP3-13739 1:100 Table 13. A list of antibodies used in assay validation. In assays with MLH1 and PMS1, antigen retrieval was tested for 30 seconds. E. Cell culture conditions - U2OS cells

[00413] U2OS cells were acquired by intoDNA from the ATCC cell culture collection. The cells were cultured at 37 °C with 5% CO2 in T25 flasks with DMEM HG + 10% FBS + 1% penicillin-streptomycin solution (10,000 units each).

[00414] The cryopreservation procedure was performed as described below. The cell suspension was centrifuged at room temperature. Petition 870250109009, dated 11 / 27 / 2025, pp. 184 / 249 176 / 190 (1000 rpm, 5 min). Then, the medium was removed and a sterile mixture containing 60% medium, 30% FBS, and 10% DMSO was added. The mixture was gently homogenized and pipetted into cryotubes (each cryotube contains 800,000 cells). The cryotube was transferred to -80 °C in a Nalgene Mr. Frosty slow-freeze container (temperature reduction of approximately 1 °C per minute). F. Seeding and treatment protocol Seeding protocol

[00415] When cell confluence reached 90%, the cells were washed with sterile PBS (without Ca2+ and Mg2+) and incubated for 4 minutes with 1 mL of 0.25% trypsin solution with EDTA. The trypsin was inactivated with the culture medium containing FBS, and the collected cells were counted in a Burker chamber. The cells were then seeded onto coverslips in 12-well plates at the densities listed in Table 14 (including the number of passages). The cells were then left to stand for 24 hours in the incubator. ATCC Name Treatment Time Number of Passes Seeding Density (number of cells / well) U2OS 48h #4-#9 75k U2OS 24h #4-#9 120k Table 14. A list of cell seeding densities. The same densities were used for the sSTRIDE and sSTRIDE MMR samples. The number of passages corresponds to the passage performed in the intoDNA laboratory. Petition 870250109009, dated 11 / 27 / 2025, pp. 185 / 249 177 / 190 Treatment protocol

[00416] After 24 hours of rest, the operating medium was replaced with fresh medium supplemented* with 6-Thioguanine (6TG)** in DMSO and left to stand for the incubation time of 24 hours or 48 hours (the final DMSO concentration in the medium was 0.1% in each experimental condition). Under vehicle conditions, the operating medium was replaced with fresh medium supplemented with 0.1% sterile DMSO and left to stand for the same incubation time. Then, all samples were fixed with 70% ethanol gel and stored at -20 °C.

[00417] *The concentration of 6TG ranged from 250 nM to 50 μM. In each well, the concentration of the diluent (DMSO) in the medium was 0.1%.

[00418] **The 6TG stock solution was freshly prepared from the powder. The powder was suspended in sterile DMSO to a final stock concentration of 100 mM. The 6TG solution in DMSO was stored in aliquots at -20 °C in the dark. G. Reagents Reagent Manufacturer Serial Number Fetal bovine serum Gibco 16140071 DMEM HG Gibco 10313021 Penicillin-streptomycin solution (10,000 units) Gibco 15140130 Petition 870250109009, dated 11 / 27 / 2025, pp. 186 / 249 178 / 190 each) Trypsin-EDTA (0.25%), Phenol Red Gibco 25200056 PBS, pH 7.2 Gibco 20012027 Dimethyl Sulfoxide Bioreagent Thermo Scientific J66650.AD DAPI Solution Thermo Scientific 62248 6-Thioguanine (6TG) Sigma Aldrich A4882-1G Table 15. List of reagents used in cell culture procedures. H. sSTRIDE-MMR Marking (PMS2)

[00419] The marking was performed in accordance with the protocol described in Example 1. I. Results

[00420] Figure 19 shows the parental HAP1 cell line versus PMS2 KO (IF assessment of staining, Abcam antibody). Representative images show an overlap of DAPI and PMS2 signals. The absence or very weak signal in the PMS2 channel confirms the KO status of one of the cell lines.

[00421] Figure 20 shows parental HAP1 versus PMS2 KO with sSTRIDE-MMR labeling. Fluorescence foci are visible in HAP1 WT cells, while most HAP1 PMS2 KO cells do not show foci.

[00422] Figure 21 shows parental HAP1: untreated control, positive control, and negative technical controls (*treatment with 6TG 1 μM for 48 h). The analysis Petition 870250109009, dated 11 / 27 / 2025, pp. 187 / 249 Quantitative imaging (179 / 190) shows that treatment with 6TG results in an increase in the number of fluorescence foci detected, while a very low number of foci is detected in the negative control samples.

[00423] Figure 22 shows HAP1 PMS2-KO: untreated control, positive control, and negative technical controls (*treatment with 6TG 1 μM for 48 h). Quantitative image analysis shows that treatment with 6TG does not result in a significant increase in the number of fluorescence foci in HAP1 PMS2 KO cells, while a very low number of foci is detected in the negative control samples.

[00424] Figure 23 shows HAP1 WT and PMS2KO cells: untreated, positive controls, and negative technical controls. The frequency distribution histograms highlight the difference in sSTRIDE-MMR results between the cell lines.

[00425] Figure 24 shows parental HAP1: control with increased 6TG concentration. Quantitative image analysis shows that treatment with higher 6TG concentration results in a higher level of sSTRIDEMMR signals. Conclusions 1) Comparison of sSTRIDE-MMR (PMS2) results between HAP1 WT and PMS2 KO cells shows a difference. Petition 870250109009, dated 11 / 27 / 2025, pp. 188 / 249 180 / 190 significant in the number of foci detected in these cell lines. As expected, a smaller number of foci were detected in the HAP1 PMS2 KO cell line. 2) The low number of foci detected in cells HAP1 PMS2 KO confirms the specificity of the assay. 3) Treatment with 6TG results in a statistically significant increase in the number of sSTRIDE MMR foci only in HAP1 WT cells. Example 6 - Comparison of dSTRIDE, dSTRIDE-RAD51, and dSTRIDE-RPA results in NCI-H661 (i.e., RH proficient) and NCI-H1693 (i.e., RH deficient) cell lines. Cell culture conditions

[00426] NCI-H1693 and NCI-H661 cell lines were acquired from the ATCC cell culture collection (May 18, 2022). A detailed description of the cell culture is listed in Table 16. Cells were cultured at 37 °C with 5% CO2 in T75 flasks containing RPMI 1640 + 10% FBS + 1% penicillin-streptomycin solution (10,000 units each). The master bank collection was prepared from passage no. 1.

[00427] The cryopreservation procedure was performed as described below. The cell suspension was centrifuged at room temperature (1000 rpm, 5 min). Then, the medium was removed and a sterile mixture containing 60% medium, 30% Petition 870250109009, dated 11 / 27 / 2025, pp. 189 / 249 181 / 190 of FBS and 10% DMSO. The mixture was gently homogenized and pipetted into cryotubes (each cryotube containing 800,000 cells). The cryotube was transferred to -80 °C in a Nalgene Mr. Frosty slow-freezing container (temperature reduction of approximately 1 °C per minute). After 120 minutes, the cryotube was transferred to liquid nitrogen. The experiments included in this report were performed using the cell bank prepared on May 30, 2022. Cell line Origin of cell line ATCC name Lot number NCI-H1693 Human lung adenocarcinoma CRL-5887 70015981 NCI-H661 Human lung carcinoma HBT-183 70007203 Table 16. A list of cell phone banks acquired from ATCC. Seeding and treatment protocol Seeding protocol When cell confluence reached 90%, the cells were washed with sterile PBS (without Ca2+ and Mg2+) and incubated for 2 minutes with 2 mL of 0.25% trypsin solution with EDTA. The trypsin was inactivated with the culture medium containing FBS, and the collected cells were counted in a Burker chamber. Then, the cells were seeded onto coverslips in 12-well plates at the densities listed in Table 17 (also including the number of passages). The cells were Petition 870250109009, dated 11 / 27 / 2025, pp. 190 / 249 182 / 190 then left to rest for 24 hours in the incubator. ATCC Name Cell Line Passage Number Seeding Density (number of cells / well) CRL-5887 NCI-H1693 #3 80k HBT-183 NCI-H661 #5 70k Table 17. A list of cell seeding densities. The same densities were used for dSTRIDE, dSTRIDE HR, and dSTRIDE RPA in each treatment condition. Treatment protocol

[00428] After 24 hours of rest, the operating medium was replaced with fresh medium supplemented with 5 μM or 50 μM of etoposide* and left to stand for the incubation time of 48 hours (the final DMSO concentration in the medium was 0.1% in each experimental condition). Under vehicle conditions, the operating medium was replaced with fresh medium supplemented with 0.1% sterile DMSO and left to stand for the same incubation time. Then, all samples were fixed with ice-cold 70% ethanol and stored at -20 °C.

[00429] *The etoposide stock (100 mM in DMSO) is stored at -20 °C in the dark. Reagents Reagent Manufacturer Serial Number Fetal bovine serum Gibco 16140071 RPMI 1640 Gibco 11875101 Penicillin-streptomycin solution (10,000 units each) Gibco 15140130 Trypsin-EDTA (0.25%), phenol red Gibco 25200056 Petition 870250109009, dated 11 / 27 / 2025, pp. 191 / 249 183 / 190 PBS, pH 7.2 Gibco 20012027 Dimethyl sulfoxide bioreagent Thermo Scientific J66650.AD DAPI solution Thermo Scientific 62248 Etoposide Torcis Chemicals 6A / 24772 Table 18. List of reagents used in cell culture procedures. dSTRIDE / dSTRIDE-RAD51 / dSTRIDE-RPA marking

[00430] The marking was performed in accordance with the protocols described in Examples 2 or 3. Results 1) In both cell lines, treatment with etoposide resulted in a dose-dependent increase in the number of DNA double-strand breaks detected by dSTRIDE. 2) The baseline level of DSBs (dSTRIDE) and the increase after treatment with 5 μM etoposide are comparable in both cell lines. 3) Treatment with 50 μM of etoposide results in a more pronounced increase in the number of DSBs (dSTRIDE) in the NCI-H1693 cell line (i.e., RH-deficient). 4) In both cell lines, treatment with etoposide resulted in a dose-dependent increase in the number of RPA-associated double bond breaks detected by dSTRIDE-RPA. 5) Treatment with 50 μM of etoposide resulted in a significant increase in the number of double bond breaks. Petition 870250109009, dated 11 / 27 / 2025, pp. 192 / 249 184 / 190 RAD51-associated cells detected by dSTRIDE-RAD51 in the NCI-H661 cell line (i.e., RH-proficient). 6) Treatment with etoposide did not result in a detectable increase in the number of RAD51-associated double bond breaks detected by dSTRIDE-RAD51 in the NCI-H1693 cell line (i.e., RH-deficient).

[00431] Figure 25 shows dSTRIDE images with and without etoposide. Representative images show the increase in the number of DNA double breaks detected by dSTRIDE and represented as fluorescence foci in the RH-deficient strain (i.e., NCI-H1693) after treatment with etoposide.

[00432] Figure 26 shows results of the dSTRIDE signal data analysis, measurement: level of DNA double-strand breaks after etoposide treatment. Quantitative image analysis shows a dose-dependent increase in the number of DSBs after etoposide treatment.

[00433] Figure 27 shows results of the dSTRIDE signal data analysis, measurement: level of DNA double-strand breaks after treatment with etoposide. *in the vehicle condition, 5% of the most damaged cells have >192 foci.

[00434] Figure 28 shows results of the dSTRIDE signal data analysis, measuring the level of DNA double-strand breaks after treatment with etoposide. Quantitative image analysis shows a dose-dependent increase. Petition 870250109009, dated 11 / 27 / 2025, pp. 193 / 249 185 / 190 in the number of double breaks after treatment with etoposide.

[00435] Figure 29 shows results of the dSTRIDE signal data analysis, measurement: level of DNA double-strand breaks after treatment with etoposide. *in the vehicle condition, 5% of the most damaged cells have >205 foci.

[00436] Figure 30 shows results of the dSTRIDE signal data analysis, comparing cell lines. Quantitative image analysis shows that, after treatment with etoposide at 50 μM, more DSBs are induced in RH-deficient cells than in RH-proficient cells.

[00437] Figure 31 shows the IF staining of RAD51 in RH-proficient and RH-deficient cell lines. No clear difference between the cell lines is visible.

[00438] Figure 32 shows dSTRIDE-HR (RAD51) staining in an RH-proficient cell line with and without etoposide. Treatment with etoposide results in an increase in the number of visible fluorescence foci.

[00439] Figures 33 to 36 show results of the analysis of dSTRIDE HR signal data, measurement: level of double-strand breaks in DNA associated with Rad51 after treatment with etoposide. While in the RH-proficient cell line there is a dose-dependent increase in the number of dSTRIDE-HR (RAD51) foci detected after treatment with Petition 870250109009, dated 11 / 27 / 2025, pp. 194 / 249 186 / 190 etoposide, no response is visible in the RH-deficient lineage.

[00440] Figure 37 shows results of the analysis of dSTRIDE HR signal data, comparing cell lines. No response is visible in the RH-deficient cell line.

[00441] Figure 38 shows dSTRIDE-RPA staining in a RH-proficient cell line with and without etoposide. An increase in the number of foci is visible after treatment with etoposide.

[00442] Figures 39 to 42 show results of the analysis of dSTRIDE RPA signal data, measurement: level of DNA double-strand breaks associated with RPA70 after treatment with etoposide. The results show that, in both cell lines, there is a dose-dependent increase in the number of dSTRIDE-RPA foci after treatment with etoposide.

[00443] Figure 43 shows results of the analysis of dSTRIDE-RPA signal data, comparing cell lines. The results show that more dSTRIDE-RPA foci are detected in the RH-deficient cell line after treatment with 50 μM etoposide.

[00444] Figure 44 shows the comparison between dSTRIDE, dSTRIDE-HR (Rad51) and dSTRIDE-RPA in an RH-proficient cell line treated with etoposide. The Petition 870250109009, dated 11 / 27 / 2025, pp. 195 / 249 Results 187 / 190 show that there is a visible dose-dependent response in all assays after treatment with etoposide.

[00445] Figure 45 shows the comparison between dSTRIDE, dSTRIDE-HR (Rad51), and dSTRIDE-RPA in an RH-deficient cell line treated with etoposide. Quantitative image analysis revealed that etoposide treatment leads to dose-dependent formation of DNA double-strand breaks in RH-deficient cells. The dSTRIDE-RPA assay results confirm that RPA is involved in the repair of these lesions, while the dSTRIDE-HR (RAD51) assay results confirm that RAD51 recruitment to DSB sites is not efficient. Conclusions

[00446] Etoposide efficiently induces DSBs in both tested strains, but the mutation in the RH pathway in the RH-deficient strain (i.e., NCI-H1693) results in an increased level of breaks. The inability of this strain to successfully perform RH repair is clearly demonstrated by combining the dSTRIDE, dSTRIDE-RPA, and dSTRIDE-RAD51 data, since no response is observed in the latter assay, confirming that RAD51 loading is insufficient. The dSTRIDE-RAD51 assay can therefore be considered a functional RH assay that provides information on the state of homologous recombination repair. This information can be used in the method to predict whether a Petition 870250109009, dated 11 / 27 / 2025, pp. 196 / 249 188 / 190 A tumor from an individual diagnosed with cancer is capable of performing DNA repair by homologous recombination. Specifically, a tumor sample taken from the individual can be analyzed using the STRIDE protocol described in this document for the level of RAD51 and / or RAD70 proteins. If the quantity is lower than expected compared to a reference cell sample, the tumor sample may contain deficiencies in the proteins involved in RH repair. If the quantity is comparable to or higher than expected compared to a reference cell sample, the tumor sample may not contain deficiencies in the proteins involved in RH repair. This information can also be used in selecting an appropriate anticancer agent (e.g., a PARP inhibitor) for the individual's treatment.Furthermore, this information can be used in a method to predict the response of an individual diagnosed with cancer to an anticancer treatment, especially if the anticancer treatment targets proteins involved in the HR pathway (or the mismatch repair pathway). This information can additionally be used in the method to select a personalized therapy for an individual diagnosed with cancer, especially if the therapy includes molecules that target proteins involved in the HR pathway (or the mismatch repair pathway). For example, the molecule may... Petition 870250109009, dated 11 / 27 / 2025, pp. 197 / 249 189 / 190 being a PARP inhibitor. Example 7 - Development of the sSTRIDE-MMR assay (PMS1 and MLH1)

[00447] The sSTRIDE-MMR methods were performed as per Examples 1 and 5. However, PMS1 or MLH1 were targeted instead of PMS2.

[00448] Figure 46 shows results of quantitative analysis in HAP1 cells (WT and KO) for sSTRIDE-MLH1. Quantitative image analysis reveals that the MLH1 protein is present in single-strand breaks (SSBs) in both PMS2 WT and KO cell lines, and that treatment with 6TG results in an increase in sSTRIDE-MMR (MLH1) results.

[00449] Figure 47 shows results of quantitative analysis in HAP1 cells for sSTRIDE-MLH1 with or without 6TG treatment. The frequency distribution plots confirm the results of the sSTRIDE-MMR (MLH1) assay.

[00450] Figure 48 shows the comparison between the results of PMS2 and MLH1. The results show that, while PMS2 is present at very low levels in HAP1 PMS2 KO cells, MLH1 can be recruited to SSBs in both cell lines.

[00451] Figure 49 shows results of quantitative analysis in HAP1 cells (WT and KO) for sSTRIDE-PMS1. The results obtained show that PMS1 is present in SSBs in both cell lines and that it participates in the processes Petition 870250109009, dated 11 / 27 / 2025, pp. 198 / 249 190 / 190 MMR after treatment with 6TG.

[00452] Figure 50 shows the comparison between the results of PMS2, PMS1 and MLH1. The results show that both MLH1 and PMS1 are involved in MMR in the absence of PMS2. Example 8 - dSTRIDE-HR assay (RAD51 and RPA) in cancer biopsy samples

[00453] The dSTRIDE-HR methods were performed as per Examples 2 and 3 on breast cancer biopsy samples from patients (a) without BRCA mutation (i.e., wild type), (b) with BRCA1 gene mutation, and (c) with BRCA2 gene mutation. Samples were tested independently for RAD51 (as per Example 2 protocol) and RPA (as per Example 3 protocol).

[00454] The results show significantly lower levels of DNA ends bound to RAD51 and RPA in samples from patients with mutations in the BRCA1 and BRCA2 genes, compared to the levels measured in samples from patients without BRCA mutation (wild type). This indicates a deficient homologous recombination pathway in samples with mutations in the BRCA1 and BRCA2 genes. Samples with a deficient RH pathway indicate suitability for treatment with anticancer agents, for example, a PARP inhibitor.

Claims

1. Method for detecting nucleic acid end(s) in a biological material containing nucleic acids, the method characterized in that it comprises the steps of: a) incubating the biological material with a proteinase for less than 2 minutes; b) adding nucleic acid binding molecules to the biological material under conditions such that the nucleic acid binding molecules bind to the nucleic acid end(s) in the biological material; c) adding two different binding molecules to the biological material under conditions such that one of the binding molecules binds to the nucleic acid binding molecules attached to the nucleic acid end(s) and the other binding molecule binds to a nucleic acid binding protein that is attached to the nucleic acid at the nucleic acid end(s);ed) detect the nucleic acid end(s) in biological material by detecting colocalization of the nucleic acid-binding protein and the nucleic acid end through the binding of two different binding molecules.

2. Method, according to claim 1, characterized in that the nucleic acids Petition 870250109007, of 11 / 27 / 2025, p. 10 / 20 2 / 10 comprise DNA and / or RNA, preferably DNA.

3. A method according to claim 1 or claim 2, characterized in that the nucleic acid end(s) is / are a single-stranded nucleic acid break or a double-stranded nucleic acid break.

4. A method according to any one of claims 1 to 3, characterized in that the proteinase is an aspartic protease, a glutamic protease, a metalloprotease, a cysteine ​​protease, a serine protease or a threonine protease, preferably in which the protease is a serine protease, such as proteinase K.

5. A method according to any one of claims 1 to 4, characterized in that the incubation of the biological material with a proteinase is carried out: a. for 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.

6. Method, according to any one of claims 1 to 5, characterized in that the proteinase is in solution, preferably at a concentration between 1 μg / mL and 20 pg / mL, between 3 pg / mL and 15 μg / mL, between 5 pg / mL and Petition 870250109007, dated 11 / 27 / 2025, page 11 / 20 3 / 10 10 μg / mL, or between 6 μg / mL and 8 μg / mL, preferably about 7 μg / mL.

7. A method according to any one of claims 1 to 6, characterized in that the nucleic acid-binding molecules: a. are halogenated nucleotide or nucleoside molecules, DNA precursor analogs and / or biotinylated nucleotide molecules; and / or b. bind to the nucleic acid end(s) in the biological material by an enzyme-catalyzed addition process, for example, using DNA polymerase I, TdT, Klenow fragment, Phu polymerase, Taq polymerase, T4 DNA polymerase, T7 DNA polymerase, T4 polynucleotide kinase or RNA polymerase.

8. A method according to any one of claims 1 to 7, characterized in that: a. the two different binding molecules may be or may comprise two different monoclonal antibodies, or fragments thereof, and / or b. each of the two different binding molecules is linked to a different oligonucleotide molecule.

9. Method, according to any one of claims 1 to 8, characterized in that the nucleic acid-binding protein is: a. a protein found in biological material; Petition 870250109007, dated 11 / 27 / 2025, p. 12 / 20 4 / 10 b. linked to the nucleic acid at the nucleic acid end(s), directly or indirectly; c. a nucleic acid repair protein, preferably a nucleic acid end repair protein (e.g., break); and / or d.RAD51, RPA (e.g., RPA70), PMS2, MLH1, PMS1, p53, MSH2, Ataxia telangiectasia and Rad3-related protein, ATM serine / threonine kinase, RAD52, XRCC1, Proliferative cell nuclear antigen, XPC, Ku70, Ku80, Nibrin, DDB2, Bloom syndrome protein, CHEK2, RAD51C, DNA polymerase eta, Rad50, DDB1, RBBP8, FANCB, PALB2, H2AX, RAD54-like DNA repair and recombination protein yH2AX, PrimPol, REV1, Terminal deoxynucleotidyl transferase, nude DNA polymerase, 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, or PARP1 / 2.

10. Method, according to any one of claims 1 to 9, the method characterized in that it further comprises a step of adding two different additional linking molecules that bind respectively to the two different linking molecules after the step of adding the two different linking molecules, optionally wherein each of the two different additional linking molecules is linked to a different oligonucleotide molecule.

11. Method, according to any one of claims 1 to 10, the method characterized in that it further comprises, after the step of adding two different linking molecules (or after the step of adding the additional linking molecules when dependent on claim 10), a step of adding additional different oligonucleotide molecules that hybridize with the oligonucleotide molecules linked to the two different linking molecules (or to the additional linking molecules) to form a circular template, optionally, wherein the circular template is formed by linking the two additional different oligonucleotide molecules.

12. Method, according to any one of claims 1 to 11, characterized in that the detection of the nucleic acid end(s) comprises a step of performing nucleic acid amplification to produce an amplification product that is detected, optionally wherein the nucleic acid amplification is rolling circle amplification. Petition 870250109007, dated 11 / 27 / 2025, page 14 / 20 6 / 10 13. A method according to any one of claims 1 to 12, characterized in that the detection of the colocalization of nucleic acid-binding protein and nucleic acid end comprises: a. a proximity binding assay; b. a branched proximity hybridization assay; c. a FRET detection; or d. a proximity-driven reaction with fluorophores or dyes.

14. Kit for detecting nucleic acid end(s) in a biological material, the kit characterized in that it comprises: a) nucleic acid binding molecules; b) a binding molecule that binds to nucleic acid binding molecules; and c) a binding molecule that binds to a nucleic acid binding protein.

15. Method for evaluating the efficacy of a therapeutic agent, the method characterized in that it comprises the steps of: a) performing the method, as defined in any one of claims 1 to 13, on a sample obtained from an individual before administration of the therapeutic agent, b) performing the method, as defined in any one of claims 1 to 13, on a sample obtained from the individual Petition 870250109007, dated 11 / 27 / 2025, p. 15 / 20 7 / 10 after administration of the therapeutic agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), where the therapeutic agent is effective if the amount of nucleic acid end(s) detected is greater in step a) than in step b), where steps a) and b) can be performed in any order, optionally, where the therapeutic agent targets PMS1, PMS2 or MLH1;or a) perform the method, as defined in any one of claims 1 to 13, on a sample obtained from an individual before administration of the therapeutic agent, b) perform the method, as defined in any one of claims 1 to 13, on a sample obtained from the individual after administration of the therapeutic agent, c) compare the amount of nucleic acid end(s) detected in steps a) and b), wherein the therapeutic agent is effective if the amount of nucleic acid end(s) detected is greater in step b) than in step a), wherein steps a) and b) may be performed in any order, optionally, wherein the therapeutic agent is targeted by Petition 870250109007, dated 11 / 27 / 2025, page 16 / 20 8 / 10 RPA or RAD51.; 16. Use of a kit for detecting nucleic acid end(s) in a biological material, the kit characterized in that it comprises: a) nucleic acid binding molecules; b) a binding molecule that binds to nucleic acid binding molecules; and c) a binding molecule that binds to a nucleic acid binding protein.

17. Kit according to claim 14, or use according to claim 16, characterized in that the kit additionally comprises a proteinase.

18. Kit or use, according to claim 17, characterized in that the proteinase is proteinase K.

19. A method for predicting the response of an individual diagnosed with cancer to an anticancer treatment, characterized in that it comprises (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual performing the method, as defined in any one of claims 1 to 13; and (ii) predicting the individual's response to an anticancer treatment based on the determined level of the nucleic acid-binding protein in the sample.

20. Method for selecting a personalized therapy for an individual diagnosed with cancer, characterized Petition 870250109007, dated 11 / 27 / 2025, page 17 / 20 9 / 10 by the fact that it comprises (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual performing the method, as defined in any one of claims 1 to 13; and (ii) selecting a personalized therapy for the individual based on the determined level of the nucleic acid-binding protein in the sample.

21. A method for predicting whether a tumor in an individual diagnosed with cancer is capable of repairing DNA by homologous recombination, characterized in that it comprises (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual performing the method, as defined in any one of claims 1 to 13; and (ii) predicting whether a tumor in the individual diagnosed with cancer is capable of repairing DNA by homologous recombination based on the determined level of the nucleic acid-binding protein in the sample.

22. Method for evaluating the state of a mismatch repair pathway in a sample comprising a tumor cell obtained from an individual, the method characterized in that it comprises (i) determining the level of a nucleic acid-binding protein in the tumor cell by performing the method as defined in any one of claims 1 to 13; and (ii) evaluating the state of the mismatch repair pathway based on the determined level of the nucleic acid-binding protein in the tumor cell.

23. A method for classifying an individual diagnosed with cancer into a cohort of patients, characterized in that it comprises (i) determining the level of a nucleic acid-binding protein in a sample obtained from the individual performing the method, as defined in any one of claims 1 to 13; and (ii) classifying an individual diagnosed with cancer into a cohort of patients based on the determined level of the nucleic acid-binding protein in the sample.

24. Method according to any one of claims 19 to 23, characterized in that the nucleic acid binding protein is RAD51, RPA70, PMS1, PMS2 or MLH1.

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