Potassium-specific dnazymes

Abstract

Disclosed herein is a DNAzyme sensor comprising a substrate strand and an enzyme strand comprising a catalytic loop, wherein the catalytic loop is capable of cleaving the substrate strand in the presence of K+, thereby activating a detectable signal. Also disclosed herein are methods of detecting K+ using the disclosed DNAzyme sensors. Further disclosed herein are methods of determining usefulness of test agents in modulating K+ and methods of treating and / or preventing dysregulation of K+ using the disclosed DNAzyme sensors.

Description

Attorney Docket No.10046-637WO1 POTASSIUM-SPECIFIC DNAZYMES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 683,384, filed August 15, 2024, which is incorporated by reference herein in its entirety. ^ GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. R35 GM141931 awarded by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on August 15, 2025, as an .XML file entitled “10046-637WO1_ST26.xml” created on July 21, 2025, and having a file size of 36,876 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND

[0004] Cancer resistance and recurrence present critical challenges in oncology, complicating treatment and reducing the efficacy of chemotherapy and targeted therapies. The observation of elevated potassium ion (K+) concentrations within the tumor microenvironment raises important questions about their role in cancer progression and treatment resistance. Recent research has demonstrated that high K+levels in the tumor microenvironment can suppress T-cell activity, creating an immunosuppressive milieu that benefits cancer cells. Understanding whether cancer cells actively maintain high K+levels in their environment to gain survival advantages is crucial. Uncovering the role of K+in conferring anticancer drug resistance would be particularly significant. Given the multifaceted role of K+in cell apoptosis, immunology, and cancer biology, developing sensors to accurately probe K+levels and distribution in cancer cells is of great significance. These insights could lead to novel therapeutic strategies aimed at manipulating K+concentrations or K+channel activities to enhance the effectiveness of cancer treatments and potentially overcome drug resistance.

[0005] Thus, there exists a need for improved sensors and methods for monitoring K+in cells. These needs and others are at least partially satisfied by the present disclosure. ^Attorney Docket No.10046-637WO1 SUMMARY

[0006] In an aspect, provided is a DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when potassium (K+) is not present; and an enzyme strand at least partially complementary to the substrate strand and including a catalytic loop; wherein the catalytic loop is capable of cleaving the substrate strand at the cleavage site in the presence of K+, wherein said cleavage can active the detectable signal; wherein the catalytic loop can include SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof.

[0007] In another aspect, provided is a method of detecting K+, the method including: a) exposing a sample to any of the disclosed DNAzyme sensors; and b) identifying the detectable signal, thereby detecting K+in the sample.

[0008] In yet another aspect, provided is a method of determining usefulness of a test agent in modulating K+, the method including: a) exposing the test agent to a sample; b) exposing the sample to any of the disclosed DNAzyme sensors; c) identifying the detectable signal, thereby detecting K+in the sample; and d) using said detectable signal to determine an effect of the test agent on K+.

[0009] In yet still another aspect, provided is a method of treating and / or preventing dysregulation of K+in a subject in need thereof, the method including: a) administering to the subject any of the disclosed DNAzyme sensors; b) identifying the detectable signal, thereby detecting K+in the subject; and c) using said detectable signal to trigger a therapeutic event.

[0010] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims. BRIEF DESCRIPTION OF DRAWINGS

[0011] FIGURE 1A shows the predicted structure of the cis-10-9 DNAzyme (SEQ ID NO: 10). FIGURE 1B shows the structure of trans-10-9 DNAzyme (substrate strand: SEQ ID NO: 4) (enzyme strand: SEQ ID NO: 7) post sequence truncation.

[0012] FIGURE 2A shows PAGE based activity assay of the trans-10-9 DNAzyme under different K+concentrations. FIGURE 2B shows the dynamic response of the trans-20-4 DNAzyme under different K+concentrations.

[0013] FIGURES 3A-3F depict the performance of the K+DNAzyme sensor. FIG. 3A shows the structure of the active K+DNAzyme sensor (substrate strand: SEQ ID NO: 4) ^Attorney Docket No.10046-637WO1 (enzyme strand: SEQ ID NO: 7). The ribonucleotide cleavage site is rG. The of nucleotide being mutated to abolish the activity of the sensor is nucleotide 12 in SEQ ID NO: 7. FIG.3B shows the fluorescence increases of the active sensor over time at different K+concentrations. FIG. 3C shows the dynamic response of the K+DNAzyme sensor under different K+concentrations. Inset: the linear response range of the sensor. FIG. 3D shows the structure of the inactive K+DNAzyme sensor (substrate strand: SEQ ID NO: 4) (inactive enzyme strand: SEQ ID NO: 11). FIG.3E shows the fluorescence increases of the inactive sensor over time at different K+concentrations. FIG.3F shows the normalized selectivity of the sensor over other monovalent (100 mM), divalent (2 mM), and trivalent (0.2 mM) metal ions.

[0014] FIGURE 4A shows live imaging of K+in HeLa cells by active and inactive K+DNAzyme sensors. FIGURE 4B shows quantification of K+DNAzyme sensor live cell imaging data in HeLa cells.

[0015] FIGURES 5A-5D depict differential K+imaging in live cells under K+regulations. FIG. 5A shows live cell K+imaging in HeLa cells treated with ionophore and different concentrations of K+. FIG. 5B is a plot graph showing the quantification of fluorescence intensity from HeLa cells treated with ionophore and different concentrations of K+. FIG.5C shows live cell K+imaging in HeLa cells treated with K+selective chelator at different concentrations. FIG. 5D is a plot graph showing the quantification of fluorescence intensity from HeLa cells treated with different concentrations of K+selective chelator.

[0016] FIGURES 6A-6B depict intracellular imaging of K+in breast tissue cells during carcinogenesis. FIG. 6A shows live imaging of K+in breast tissue cells representing different stages of cancer progression. FIG. 6B is a plot graph show the quantification of fluorescence intensity from breast tissue cells.

[0017] FIGURE 7 depicts live imaging of K+in breast tissue cells representing different stages of cancer progression by inactive K+DNAzyme sensors.

[0018] FIGURES 8A-8D depict the resistance of ML133 molecule in MDA-MB-231 cells under different environmental K+conditions. FIG. 8A shows live imaging of SYTO 82 / SYTOX Green staining of MDA-MB-231 cells under different environmental K+conditions and ML133 drug concentrations. FIG. 8B is a bar graph showing the quantification of fluorescence intensity from MTT assays. FIG. 8C is a plot graph showing the quantification of fluorescence intensity under different concentrations of ML133 treatment by the K+sensor. FIG. 8D is a plot graph showing the quantification of fluorescence intensity with or without ML133 treatment in media with different K+levels by the K+sensor. ^Attorney Docket No.10046-637WO1

[0019] FIGURES 9A-9D depict the resistance of Amiodarone molecule in MDA-MB-231 cells under different environmental K+conditions. FIG.9A shows live imaging of SYTO 82 / SYTOX Green staining of MDA-MB-231 cells under different environmental K+conditions and Amiodarone drug concentrations. FIG. 9B is a bar graph showing the quantification of fluorescence intensity from MTT assays. FIG. 9C is a plot graph showing the quantification of fluorescence intensity under different concentrations of Amiodarone treatment by the K+sensor. FIG. 9D is a plot graph showing the quantification of fluorescence intensity with or without Amiodarone treatment in media with different K+levels by the K+sensor.

[0020] FIGURES 10A-10G depict performance of the K+DNAzyme sensor. FIG.10A is a scheme showing the molecular mechanism by which K+activates the DNAzyme sensor (substrate strand: SEQ ID NO: 4) (enzyme strand: SEQ ID NO: 7) and generates a fluorescent signal. The ribonucleotide cleavage site is rG. FIG. 10B shows fluorescence increases of the active sensor over time at different K+concentrations. FIG.10C shows the dynamic response of the K+DNAzyme sensor under different K+concentrations. FIG. 10D shows the linear response range of the sensor. FIG.10E shows the structure of the inactive K+DNAzyme sensor (substrate strand: SEQ ID NO: 4) (inactive enzyme strand: SEQ ID NO: 11). The mutation abolishing the activity of the sensor is at position nucleotide 12 in SEQ ID NO: 7. FIG. 10F shows fluorescence increases of the inactive sensor over time at different K+concentrations. FIG. 10G shows normalized activity of the K+sensor over other monovalent (100 mM), divalent (2 mM), and trivalent (0.2 mM) metal ions at concentrations consistent with a prior report of monovalent DNAzyme selection studies

[0062] . The in vitro fluorescence assays were tested in 50 mM Tris-HCl buffer at pH 7.4.

[0021] FIGURE 11 depicts quantification of intracellular K+imaging (via intracellular fluorescence intensity) by the DNAzyme sensor from live cell imaging data in HeLa cells. **** p < 0.0001.

[0022] FIGURES 12A-12D depict K+imaging in live cells under different K+regulations. FIG. 12A shows live cell K+imaging in HeLa cells treated with ionophore and different concentrations of K+. FIG. 12B is a plot graph showing the quantification of fluorescence intensity from HeLa cells treated with ionophore and different concentrations of K+. FIG.12C shows live cell K+imaging in HeLa cells treated with K+selective chelator at different concentrations. FIG. 12D is a plot graph showing the quantification of fluorescence intensity from HeLa cells treated with different concentrations of K+selective chelator. Cells were transfected for 4 hours, followed by a 30-minute wash and incubation prior to imaging. Scale bar: 50 µm. Two-tailed unpaired t-test: ns p > 0.05, * p < 0.05, ** p < 0.01, **** p<0.0001. ^Attorney Docket No.10046-637WO1

[0023] FIGURES 13A-13C depict intracellular imaging of K+in breast tissue cells during carcinogenesis. FIG.13A shows live imaging of K+in breast tissue cells representing different stages of cancer progression. FIG. 13B is a plot graph showing the quantification of fluorescence intensity from breast tissue cells. FIG. 13C shows ICP-MS analysis demonstrating the K+exportation from breast tissue cells. Scale bar: 20 µm. Two-tailed unpaired t-test: * p < 0.05, *** p < 0.001, **** p<0.0001.

[0024] FIGURE 14 depicts ICP-MS analysis of intracellular K+levels in breast tissue cells representing different stages of cancer progression.

[0025] FIGURES 15A-15D depict resistance of ML133 treatment in MDA-MB-231 cells under different environmental K+conditions. FIG. 15A shows a cell viability assay of MDA- MB-231 cells under different environmental K+conditions and ML133 concentrations. FIGS. 15B-15C show fluorescent imaging (FIG.15B) and quantification (FIG.15C) of K+imaging in MDA-MB-231 cells treated by ML133 at different concentrations by the K+sensor. FIGS. 15D-15E show fluorescent imaging (FIG.15D) and quantification (FIG.15E) of K+imaging in MDA-MB-231 cells treated with different environmental K+and ML133 combinations by the K+sensor. Scale bar: 100 µm. Two-tailed unpaired t-test: ** p < 0.01, **** p<0.0001.

[0026] FIGURES 16A-16H depict resistance of Amiodarone treatment in MDA-MB-231 cells under different environmental K+conditions. FIG. 16A shows a cell viability assay of MDA-MB-231 cells under different environmental K+conditions and Amiodarone concentrations. FIGS. 16B-16C show fluorescent imaging (FIG. 16B) and quantification (FIG. 16C) of K+imaging in MDA-MB-231 cells treated by Amiodarone at different concentrations by the K+sensor. FIGS. 16D-16E show fluorescent imaging (FIG. 16D) and quantification (FIG. 16E) of K+imaging in MDA-MB-231 cells treated with different environmental K+and Amiodarone combinations by the K+sensor. Scale bar: 100 µm. FIGS. 16F-16H shows fluorescent imaging (FIG.16F), cell viability (FIG.16G), and quantification of K+imaging (FIG. 16H) in MDA-MB-231 cotreated by Amiodarone and ML133 under different media K+levels. Scale bar: 50 µm. Two-tailed unpaired t-test: ns p > 0.05, ** p < 0.01, *** p < 0.001, **** p<0.0001. DETAILED DESCRIPTION

[0027] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. ^Attorney Docket No.10046-637WO1 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. DEFINITIONS

[0028] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0029] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0030] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0031] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0032] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater ^Attorney Docket No.10046-637WO1 than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0033] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub- ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0034] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0035] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve ^Attorney Docket No.10046-637WO1 the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0036] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0037] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0038] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or ^Attorney Docket No.10046-637WO1 medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0039] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0040] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0041] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0042] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0043] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. ^Attorney Docket No.10046-637WO1 Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0044] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0045] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0046] Reference is made herein to nucleic acid and nucleic acid sequences. The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single- stranded or double-stranded and may represent the sense or the antisense strand).

[0047] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5^-terminal or 3^-terminal truncation or both of a reference polynucleotide).

[0048] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide ^Attorney Docket No.10046-637WO1 or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C-terminal region of a polypeptide or the 5^-terminal region and / or the 3^ terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.

[0049] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0050] Fusion polynucleotides also are contemplated herein. A “fusion polynucleotide” refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3^ end of a first polynucleotide to a 5^ end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0051] “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences or polynucleotide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.

[0052] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local ^Attorney Docket No.10046-637WO1 Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0053] Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0054] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0055] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide. A variant polynucleotide may have substantially the same functional activity as a reference polynucleotide.

[0056] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. ^Attorney Docket No.10046-637WO1

[0057] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0058] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 13, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0059] “Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0060] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

[0061] The term “mismatched” or “mismatched target sequence” refers to an off-target sequence that is not perfectly complementary to the first DNA sequence or the second DNA sequence of the chimeric deoxyribonucleic acid described herein. The dual retargeted DNA ^Attorney Docket No.10046-637WO1 may have at least one mismatch, but can also have 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides to the off-target sequence.

[0062] As used herein, the term “detecting” used in context of detecting a signal from a detectable label or detectable signal to indicate the presence of a target nucleic acid (such as a signaling nucleic acid) in the sample does not require the method to provide 100% sensitivity and / or 100% specificity. As is well known, “sensitivity” is the probability that a test is positive, given that the sample has a target nucleic acid sequence, while “specificity” is the probability that a test is negative, given that the sample does not have the target nucleic acid sequence. A sensitivity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. A specificity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. Detecting also encompasses assays with false positives and false negatives. False negative rates may be 1%, 5%, 10%, 15%, 20% or even higher. False positive rates may be 1%, 5%, 10%, 15%, 20% or even higher. The term “detecting” is also used in the context of detecting the amplified target nucleic acid by its melting temperature using melting curve analysis, as is known in the art.

[0063] As used herein, “detectable labels” or “detectable signals” are chemical or biochemical moieties useful for labeling a nucleic acid (including a single nucleotide), amino acid, or antibody. “Detectable labels” or “detectable signals” include fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionuclides, enzymes, substrates, cofactors, inhibitors, magnetic particles, quantum dots, and other moieties known in the art. “Detectable labels” or “detectable signals” are capable of generating a measurable signal, or can be used to capture nucleic acids, and may be covalently or noncovalently joined to an oligonucleotide or nucleotide (e.g., a non-natural nucleotide).

[0064] As used herein, the term “DNAzymes,” also called “deoxyribozymes,” are DNA molecules that display enzymatic activities, such as protein enzymes and ribozymes, in the presence of a cofactor such as metal ions or another target molecule. DNAZYMES

[0065] In an aspect, provided is a DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when potassium (K+) is not present; and an enzyme strand at least partially complementary to the substrate strand and including a catalytic loop; wherein the catalytic loop is capable of cleaving the substrate strand at the cleavage site in the presence of K+, wherein said cleavage can active ^Attorney Docket No.10046-637WO1 the detectable signal; wherein the catalytic loop can include SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof. TABLE 1. DNAzyme sequences.^Attorney Docket No.10046-637WO1

[0066] In some aspects, the cleavage site can include 1 or more RNA bases (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more). In some aspects, the cleavage site can include 25 or less RNA bases (e.g., 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less).

[0067] The cleavage site can include any number of RNA bases ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the cleavage site can include from 1 to 25 RNA bases (e.g., from 2 to 24, from 3 to 23, from 4 to 22, from 5 to 21, from 6 to 20, from 7 to 19, from 8 to 18, from 9 to 17, from 10 to 16, from 11 to 15, from 12 to 14, from 1 to 13, from 2 to 12, from 3 to 11, from 4 to 10, from 5 to 9, from 6 to 8, from 13 to 25, from 14 to 24, from 15 to 23, from 16 to 22, from 17 to 21, from 18 to 20).

[0068] In some aspects, about 50% or more (e.g., 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100%) of the nucleotides in the substrate strand can be RNA bases. In some aspects, all of the nucleotides in the substrate strand can be RNA bases.

[0069] In some aspects, when more than one RNA base is present, all RNA bases may be immediately adjacent. In other aspects, when more than one RNA base is present, at least one RNA base may be distanced from the other RNA bases by at least one other nucleotide. ^Attorney Docket No.10046-637WO1

[0070] In some aspects, the cleavage site can be interspersed between two segments of nucleic acid. In some such aspects, the two segments of nucleic acid can have a same length. In other such aspects, the two segments of nucleic acid can have different lengths.

[0071] In some aspects, each of the two segments of nucleic acid can include at least 3 nucleotides (e.g., at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides). In some aspects, each of the two segments of nucleic acid can include up to 30 nucleotides (e.g., up to 29 nucleotides, up to 28 nucleotides, up to 27 nucleotides, up to 26 nucleotides, up to 25 nucleotides, up to 24 nucleotides, up to 23 nucleotides, up to 22 nucleotides, up to 21 nucleotides, up to 20 nucleotides, up to 19 nucleotides, up to 18 nucleotides, up to 17 nucleotides, up to 16 nucleotides, up to 15 nucleotides, up to 14 nucleotides, up to 13 nucleotides, up to 12 nucleotides, up to 11 nucleotides, up to 10 nucleotides, up to 9 nucleotides, up to 8 nucleotides, up to 7 nucleotides, up to 6 nucleotides, up to 5 nucleotides, up to 4 nucleotides, up to 3 nucleotides).

[0072] It is considered that each of the two segments of nucleic acid can include a number of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, each of the two segments of nucleic acid can include from 3 nucleotides to 30 nucleotides (e.g., from 4 nucleotides to 29 nucleotides, from 5 nucleotides to 28 nucleotides, from 6 nucleotides to 27 nucleotides, from 7 nucleotides to 26 nucleotides, from 8 nucleotides to 25 nucleotides, from 9 nucleotides to 24 nucleotides, from 10 nucleotides to 23 nucleotides, from 11 nucleotides to 22 nucleotides, from 12 nucleotides to 21 nucleotides, from 13 nucleotides to 20 nucleotides, from 14 nucleotides to 19 nucleotides, from 15 nucleotides to 18 nucleotides, from 16 nucleotides to 17 nucleotides, from 3 nucleotides to 17 nucleotides, from 4 nucleotides to 16 nucleotides, from 5 nucleotides to 15 nucleotides, from 6 nucleotides to 14 nucleotides, from 7 nucleotides to 13 nucleotides, from 8 nucleotides to 12 nucleotides, from 9 nucleotides to 11 nucleotides, from 15 nucleotides to 30 nucleotides, from 16 nucleotides to 29 nucleotides, from 17 nucleotides to 28 nucleotides, from 18 nucleotides to 27 nucleotides, from 19 nucleotides to 26 nucleotides, from 20 ^Attorney Docket No.10046-637WO1 nucleotides to 25 nucleotides, from 21 nucleotides to 24 nucleotides, from 22 nucleotides to 23 nucleotides).

[0073] In some aspects, the substrate strand and / or the enzyme strand can include DNA. In some aspects, the substrate strand can further include at least one non-natural nucleic acid. For example, in some such aspects, the at least one non-natural nucleic acid can be a locked nucleic acid (LNA) or a 2’-fluoro arabino nucleic acid (FANA).

[0074] In some aspects, the enzyme strand can include a K+binding region.

[0075] In some aspects, the catalytic loop can include SEQ ID NO: 1. In some aspects, the catalytic loop can include a variant of SEQ ID NO: 1. In some aspects, the catalytic loop can include SEQ ID NO: 2. In some aspects, the catalytic loop can include a variant of SEQ ID NO: 2.

[0076] In some aspects, the substrate strand can include SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof, and the enzyme strand can include SEQ ID NO: 6, SEQ ID NO: 7, or a variant thereof. In some aspects, the substrate strand can include SEQ ID NO: 3. In some aspects, the substrate strand can include a variant of SEQ ID NO: 3. In some aspects, the substrate strand can include SEQ ID NO: 4. In some aspects, the substrate strand can include a variant of SEQ ID NO: 4. In some aspects, the enzyme strand can include SEQ ID NO: 6. In some aspects, the enzyme strand can include a variant of SEQ ID NO: 6. In some aspects, the enzyme strand can include SEQ ID NO: 7. In some aspects, the enzyme strand can include a variant of SEQ ID NO: 7.

[0077] In some aspects, the substrate strand and the enzyme strand can be linked together. In some such aspects, the substrate strand and the enzyme strand can be directly linked together. In other such aspects, the substrate strand and the enzyme strand can be linked together by a linker.

[0078] In some aspects, the linker can include nucleic acid, peptide, polymer, or any combination thereof. For example, in some aspects, the linker can include at least 1 nucleotide (e.g., at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides). In some aspects, the linker can include up to 10 nucleotides (e.g., up to 9 nucleotides, up to 8 nucleotides, up to 7 nucleotides, up to 6 nucleotides, up to 5 nucleotides, up to 4 nucleotides, up to 3 nucleotides, up to 2 nucleotides, up to 1 nucleotide).

[0079] It is considered that the linker can include any number of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the linker can include from 1 nucleotide to 10 nucleotides (e.g., ^Attorney Docket No.10046-637WO1 from 2 nucleotides to 9 nucleotides, from 3 nucleotides to 8 nucleotides, from 4 nucleotides to 7 nucleotides, from 5 nucleotides to 6 nucleotides, from 1 nucleotide to 6 nucleotides, from 2 nucleotides to 5 nucleotides, from 3 nucleotides to 4 nucleotides, from 5 nucleotides to 10 nucleotides, from 6 nucleotides to 9 nucleotides, from 7 nucleotides to 8 nucleotides.)

[0080] In some aspects, the linker can include SEQ ID NO: 9. In some aspects, the linker can include a variant of SEQ ID NO: 9.

[0081] In some aspects, the DNAzyme can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to SEQ ID NO: 10. In some aspects, the DNAzyme sensor can include SEQ ID NO: 10

[0082] In some aspects, the detectable signal can be a fluorophore or a fluorescent dye. In some such aspects, the fluorophore or fluorescent dye can be, but is not limited to Hydroxycoumarin, Alexa fluor, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer yellow, Alexa fluor 430, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, Cy2, TruRed, FluorX, Fluorescein, FAM, BODIPY-FL, TET, Alexa fluor 532, HEX, TRITC, Cy3, TMR, Alexa fluor 546, Alexa fluor 555, Tamara, X-Rhodamine, Lissamine Rhodamine B, ROX, Alexa fluor 568, Cy3.5 581, Texas Red, Alexa fluor 594, Alexa fluor 633, LC red 640, Allophycocyanin (APC), Alexa fluor 633, APC-Cy7 conjugates, Cy5, Cy5.5, LC red 705, Cy7, IRDye 800 CW, IRDye 700, Cy7.5, Dy780, Dy781, DyLight 800, IRDye 800 CW, Alexa Fluor 647, Alexa Fluor 488, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 750, Alexa Fluor 790, JOE, or MAX.

[0083] In some aspects, the detectable signal can be a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal can be activated upon exposure to an acoustic signal. In some such aspects, the detectable signal can be indocyanine green, methylene blue, Evans blue, trypan blue, patent blue, IRDye800CW, DiR, Cy7, Cy7.5, or porphyrin.

[0084] In some aspects, the detectable signal can be conjugated to a first end of the substrate strand, and a quencher can be conjugated to a complementary end of the enzyme strand. In other aspects, the detectable signal can be conjugated to a first end of the substrate strand, and a quencher can be conjugated to a second end of the substrate strand. In yet other aspects, the detectable signal can be conjugated to a first end of the substrate strand, a first ^Attorney Docket No.10046-637WO1 quencher can be conjugated to a complementary end of the enzyme strand, and a second quencher can be conjugated to a second end of the substrate strand.

[0085] In some aspects, the quencher can be, but is not limited to, DQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, BHQ-3, IRDye QC-1, or ZEN. In some aspects, when more than one quencher is present, each quencher can be the same or different.

[0086] In some aspects, the catalytic loop can have a dissociation constant (Kd) for K+of at least about 50 µM (e.g., at least about 75 µM, at least about 100 µM, at least about 150 µM, at least about 200 µM, at least about 250 µM, at least about 300 µM, at least about 350 µM, at least about 400 µM, at least about 450 µM, at least about 500 µM, at least about 600 µM, at least about 700 µM, at least about 800 µM, at least about 900 µM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 110 mM, at least about 120 mM, at least about 130 mM, at least about 140 mM, at least about 150 mM, at least about 160 mM, at least about 170 mM, at least about 180 mM, at least about 190 mM, at least about 200 mM.

[0087] In some aspects, the catalytic loop can have a dissociation constant (Kd) for K+of up to about 200 mM (e.g., up to about 190 mM, up to about 180 mM, up to about 170 mM, up to about 160 mM, up to about 150 mM, up to about 140 mM, up to about 130 mM, up to about 120 mM, up to about 110 mM, up to about 100 mM, up to about 90 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM, up to about 40 mM, up to about 30 mM, up to about 20 mM, up to about 10 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM, up to about 1 mM, up to about 900 µM, up to about 800 µM, up to about 700 µM, up to about 600 µM, up to about 500 µM, up to about 450 µM, up to about 400 µM, up to about 350 µM, up to about 300 µM, up to about 250 µM, up to about 200 µM, up to about 150 µM, up to about 100 µM, up to about 75 µM, up to about 50 µM).

[0088] It is considered that the catalytic loop can have a dissociation constant (Kd) for K+ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the catalytic loop can have a dissociation constant (Kd) for K+of from about 50 µM to about 200 mM (e.g., from about 75 µM to about 190 mM, from about 100 µM to about 180 mM, from about 150 µM to about 170 mM, from about 200 µM to about 160 mM, from about 250 µM to about 150 mM, from about 300 µM to about 140 mM, from about 350 µM to about 130 mM, from about 400 µM to about 120 mM, ^Attorney Docket No.10046-637WO1 from about 450 µM to about 110 mM, from about 500 µM to about 100 mM, from about 600 µM to about 90 mM, from about 700 µM to about 80 mM, from about 800 µM to about 70 mM, from about 900 µM to about 60 mM, from about 1 mM to about 50 mM, from about 2 mM to about 40 mM, from about 3 mM to about 30 mM, from about 4 mM to about 20 mM, from about 5 mM to about 10 mM, from about 50 µM to about 10 mM, from about 75 µM to about 5 mM, from about 100 µM to about 4 mM, from about 150 µM to about 3 mM, from about 200 µM to about 2 mM, from about 250 µM to about 1 mM, from about 300 µM to about 900 µM, from about 350 µM to about 800 µM, from about 400 µM to about 700 µM, from about 450 µM to about 600 µM, from about 5 mM to about 200 mM, from about 10 mM to about 190 mM, from about 20 mM to about 180 mM, from about 30 mM to about 170 mM, from about 40 mM to about 160 mM, from about 50 mM to about 150 mM, from about 60 mM to about 140 mM, from about 70 mM to about 130 mM, from about 80 mM to about 120 mM, from about 90 mM to about 110 mM). METHODS

[0089] In an aspect, provided is a method of detecting K+, the method including: a) exposing a sample to any of the disclosed DNAzyme sensors; and b) identifying the detectable signal, thereby detecting K+in the sample.

[0090] In some aspects, the method can further include, before step a), providing a reference level of the detectable signal by: i) providing to the sample an inactive DNAzyme sensor, the inactive DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; and an inactive enzyme strand at least partially complementary to the substrate strand including at least one mutation; wherein the at least one mutation can prevent the inactive enzyme strand from cleaving the substrate strand; and ii) identifying the detectable signal, thereby detecting K+in the sample; wherein the reference level of the detectable signal can be used to eliminate background noise in data obtained in step b).

[0091] In some aspects, the substrate strand can include SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof; and the inactive enzyme strand can include SEQ ID NO: 11 or a variant thereof. In some aspects, the substrate strand can include SEQ ID NO: 3. In some aspects, the substrate strand can include a variant of SEQ ID NO: 3. In some aspects, the substrate strand can include SEQ ID NO: 4. In some aspects, the substrate strand can include a variant of SEQ ID NO: 4. In some aspects, the inactive enzyme strand can include SEQ ID NO: 11. In some aspects, the inactive enzyme strand can include a variant of SEQ ID NO: 11.

[0092] In some aspects, the method can further include, before step a), annealing the substrate strand and the enzyme strand together. For example, in some specific aspects, the ^Attorney Docket No.10046-637WO1 substrate strand and the enzyme strand can be mixed together in 100 mM Tris-HCl, pH 7.4 in a ratio of 1:1, and incubated at 37°C for 20 minutes.

[0093] In some aspects, the substrate strand and the enzyme strand can be incubated together for at least about 5 minutes (e.g., at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes). In some aspects, the substrate strand and the enzyme strand can be incubated together for up to about 60 minutes (e.g., up to about 55 minutes, up to about 50 minutes, up to about 45 minutes, up to about 40 minutes, up to about 35 minutes, up to about 30 minutes, up to about 25 minutes, up to about 20 minutes, up to about 15 minutes, up to about 10 minutes, up to about 5 minutes).

[0094] It is considered that the substrate strand and the enzyme strand can be incubated together for a duration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the substrate strand and the enzyme strand can be incubated together for from about 5 minutes to about 60 minutes (e.g., from about 10 minutes to about 55 minutes, from about 15 minutes to about 50 minutes, from about 20 minutes to about 45 minutes, from about 25 minutes to about 40 minutes, from about 30 minutes to about 35 minutes, from about 5 minutes to about 35 minutes, from about 10 minutes to about 30 minutes, from about 15 minutes to about 25 minutes, from about 30 minutes to about 60 minutes, from about 35 minutes to about 55 minutes, from about 40 minutes to about 50 minutes).

[0095] In some aspects, the method can further include, after step a) and before step b), exposing the sample to an acoustic signal; wherein the detectable signal can be a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal can be activated upon exposure to the acoustic signal. In some aspects, the acoustic signal can be high frequency ultrasound (HIFU).

[0096] In some aspects, the method can be used to quantify K+in the sample.

[0097] In some aspects, the sample can be a cell or tissue sample. In some such aspects, the method can further include spatially identifying K+in the cell or tissue sample (i.e., intracellularly or extracellularly). In other aspects, the sample can be cell lysate or a biological fluid (e.g., sweat, urine, blood, etc.).

[0098] In some aspects, the sample can be derived from a subject having hyperkalemia or hypokalemia, or the subject may have been given medication which causes hyperkalemia or hypokalemia. In some aspects, the sample can be derived from a subject having a cancer, an ^Attorney Docket No.10046-637WO1 immune disorder, a neural disorder, a cardiovascular disorder, a kidney disorder, a metabolic disorder, or an endocrine disorder.

[0099] In some aspects, step b) can include imaging the sample and / or analyzing the sample with a fluorometer.

[0100] In some aspects, the method can be carried out in vivo, ex vivo, or in vitro.

[0101] In another aspect, provided is a method of determining usefulness of a test agent in modulating K+, the method including: a) exposing the test agent to a sample; b) exposing the sample to any of the disclosed DNAzyme sensors; c) identifying the detectable signal, thereby detecting K+in the sample; and d) using said detectable signal to determine an effect of the test agent on K+.

[0102] In some aspects, the effect of the test agent on K+can include an increase in K+amount or activity, and the detectable signal can be greater than a reference signal produced by the sample not exposed to the test agent. In other aspects, the effect of the test agent on K+can include a decrease in K+amount or activity, and the detectable signal can be lesser than a reference signal produced by the sample not exposed to the test agent. In yet other aspects, the effect of the test agent on K+can include substantially no change in K+amount or activity, and the detectable signal can be substantially the same as a reference signal produced by the sample not exposed to the test agent.

[0103] In some aspects, the sample can be a cell or tissue sample. In some such aspects, the method can further include spatially identifying K+in the cell or tissue sample (i.e., intracellularly or extracellularly). In other aspects, the sample can be cell lysate or a biological fluid (e.g., sweat, urine, blood, etc.).

[0104] In some aspects, the test agent can be a therapeutic agent or a treatment protocol. For example, in some such aspects, the test agent can be an anti-cancer agent, a beta-adrenergic agonist, a beta-blockade, an alpha-agonist, a blood pressure medication, a diuretic, an anti- inflammatory agent, an antibiotic, or a blood thinner. Additionally or alternatively, in some aspects, the test agent can physically or chemically damage the sample. For example, in some such aspects, the test agent can include ablation, scraping, exposure to radiation, and / or exposure to chemical agents (e.g., chemotherapy agents, chemical irritants, etc.).

[0105] In some aspects, the test agent can be used to treat hyperkalemia or hypokalemia. In some aspects, the test agent can induce hyperkalemia or hypokalemia.

[0106] In some aspects, the test agent can be used to treat a cancer, an immune disorder, a neural disorder, a cardiovascular disorder, a kidney disorder, a metabolic disorder, or an endocrine disorder. ^Attorney Docket No.10046-637WO1

[0107] In some aspects, the sample can be derived from a subject having hyperkalemia or hypokalemia, or the subject may have been given medication which causes hyperkalemia or hypokalemia.

[0108] In some aspects, the sample can be derived from a subject having a cancer, an immune disorder, a neural disorder, a cardiovascular disorder, a kidney disorder, a metabolic disorder, or an endocrine disorder.

[0109] In some aspects, step c) can include imaging the sample and / or analyzing the sample with a fluorometer.

[0110] In some aspects, the method can be carried out in vivo, ex vivo, or in vitro.

[0111] In yet another aspect, provided is a method of treating and / or preventing dysregulation of K+in a subject in need thereof, the method including: a) administering to the subject any of the disclosed DNAzyme sensors; b) identifying the detectable signal, thereby detecting K+in the subject; and c) using said detectable signal to trigger a therapeutic event.

[0112] In some aspects, the subject can have hyperkalemia or hypokalemia, or the subject may have been given medication which causes hyperkalemia or hypokalemia.

[0113] In some aspects, the subject can have a cancer, an immune disorder, a neural disorder, a cardiovascular disorder, a kidney disorder, a metabolic disorder, or an endocrine disorder.

[0114] In some aspects, the detectable signal can be used to determine timing, dosage, or administration route of a therapeutic agent.

[0115] In some aspects, the detectable signal can be identified by imaging the patient.

[0116] In some aspects, the detectable signal can be identified in a sample of a biological fluid (e.g., sweat, urine, blood, etc.) collected from the patient.

[0117] In some aspects, the therapeutic agent can include a small molecule, a biologic agent, a peptide, a nucleic acid, radiation, chemotherapy, and / or surgery. EXAMPLES Example 1: DNAzyme-Based Potassium-Selective Imaging Reveals Intracellular Potassium Alteration in Cancer Metastasis and Immune Cell Maturation

[0118] With the immense impact, intensive research has been performed on developing K+imaging in living cells. Among these sensory probes, small molecule-based imaging probe is the most broadly studied . However, a number of this type of K+sensors have good performance mainly in narrow pH range and small relative signal change. The majority of small molecule K+sensors and small molecule-containing nanoparticle sensors that function under physiological conditions necessitate advanced chemical and nanomaterial syntheses , making ^Attorney Docket No.10046-637WO1 them less readily accessible to researchers. The commercially available small molecule K+sensors, on the other hand, share the major limitation of low selectivity of K+over other biorelevant metal ions, especially Na+. To overcome the limitation, genetically encoded indicators based on naturally existing Escherichia coli K+binding protein (Kbp) have been successfully developed for highly selective K+detection. The early versions of the genetically encoded K+sensors (KIRIN1, KIRIN1-GR, GINKO1, GEPIIs ) have Kdor EC50less than 1 mM for K+binding and are robust tools for imaging of K+at micromolar and low millimolar concentration range. Further engineering of the genetically encoded K+sensors screened out a series of mutation carrying sensors for K+imaging at moderately higher concentration range, with the GINKO2 sensor showing Kd at 15.3 mM and lc-LysM GEPII 1.0 showing EC50 at 27.43 mM as the current best versions and have been elegantly tested in intracellular K+imaging. Genetically encoded sensors with Kd further close to the average cellular levels of K+(140-150 mM) will be ideal and expected for future research. DNA based sensors have the intrinsic advantages on biocompatibility, stability, and smaller size compared with protein coding plasmid for delivery. A few DNA based K+sensors have been reported taking advantages of DNA sequences that may fold into G-rich quadruplex or triplex structures in the presence of K+(e.g., the thrombin binding aptamers ). However, these type of K+sensors share the same limitation of suboptimal detection range at micromolar to low millimolar window and high background in detection.

[0119] DNAzyme-based metal ion sensors are a general platform for developing highly selective and biocompatible metal ion sensors. First discovered in 1994, DNAzymes are a class of DNA molecules that, when loaded with cofactors, display enzymatic activities, and metal ions are the most frequently used cofactors for DNAzyme catalyzed reactions . Among DNAzymes, RNA-cleaving DNAzymes are of particular interest for metal ion sensing due to their fast reaction rate, high metal ion cofactor selectivity, and ease of converting the cleavage into a detectable signal by the catalytic beacon strategy. Through a combinatorial strategy named “in vitro selection,” DNAzymes that are highly selective can be isolated by introducing negative or counter selection against competing metal ions, that are fast and active by adjusting the incubation time of positive selection, and most importantly, that are suitable to certain concentration dynamic range by tuning target metal ion concentrations in positive and counter selections. Using this strategy, many metal ion-specific DNAzymes have been turned into fluorescent sensors, including Zn2+, Pb2+, UO22+, Mg2+, Ca2+, Co2+, Cu2+, Cd2+, Hg2+, Ag+, Na+, Li+, Cr3+, and lanthanides, showcasing the generalizability of this strategy in generating highly selective and tunable metal ions sensors. Many of these sensors have been further engineered ^Attorney Docket No.10046-637WO1 for imaging metal ions in living cells and organisms. However, no K+selective DNAzyme with substantial activity has been reported.

[0120] This study describes the selection and characterization of an RNA-cleaving DNAzyme with high selectivity (> 1000-fold) for K+over other competing ions, with a broad detection range up to 200 mM and an estimated Kd of around 105 mM, covering the average cellular K+level of approximately 150 mM. This K+-specific DNAzyme was transformed into a biocompatible fluorescent sensor capable of detecting intracellular K+levels in living cells. The study established this sensing system for visualizing K+distribution across human breast cancer cell lines at different cancer stages to determine differential K+accumulation during metastasis. These findings revealed lower K+accumulation in metastatic breast cancer cells compared to normal states and demonstrated that high environmental K+attenuates anticancer drug efficiency. This work opens new avenues for selectively detecting K+in living cancer cells and provides opportunities to further study the physiological responses in cancer biology and tumor microenvironments. Methods and Materials

[0121] Materials: All DNA sequences were purchased from Integrated DNA Technologies (IDT) with standard desalting. Prior to use each sequence was purified via denaturing PAGE. Acrylamide / bisacrylamide 40 % solution (29:1) was obtained from Bio-Rad Laboratories, Inc. The following enzymes, reaction buffers, and reagents used were purchased from New England Biolabs: Taq polymerase, standard Taq buffer, T4-polynucleotide kinase, polynucleotide kinase buffer, and deoxynucleotide (dNTP) solution mix. Both32P labeled ^-ATP and ^-ATP were obtained from Perkin-Elmer. All PCR and32P-labeling experiments were carried out in the BioRad thermocycler.

[0122] The following chemicals were as obtained as follows: 3-(N- morpholino)propanesulfonic acid (MOPS) (Amersham International plc), Urea (Affymetrix, MB grade), Tris (Affymetrix, MB grade), boric acid (Fischer Scientific, electrophoresis grade), Ethylenediaminetetraacetic acid (EDTA) (Fluka, 99.0%), EDTA^2Na^2H2O (Fisher Scientific), 200 proof ethanol (Decon Laboratories, Inc.), sodium acetate, HCl (Alfa-Aesar, ultrapure), lithium hydroxide (Alfa aesar 99.999% puratonic salts), potassium hydroxide, sodium hydroxide, HCl (Alfa-Aesar, ultrapure). All of the metal salts used were obtained as listed: LiCl (Alfa aesar 99.999% puratonic salts), NaCl (Alfa aesar 99.999% puratonic salts), KCl (Alfa aesar 99.999% puratonic salts), CsCl (Alfa aesar 99.999% puratonic salts), RbCl (Alfa aesar 99.999% puratonic salts), MgCl2(Alfa aesar 99.999% puratonic salts), CaCl2(Alfa aesar 99.999% puratonic salts), PbCl2 (Alfa aesar 99.999% puratonic salts), MnCl2 (Alfa aesar ^Attorney Docket No.10046-637WO1 99.999% puratonic salts), SrCl2 (Alfa aesar 99.999% puratonic salts), NiCl2 (Alfa aesar 99.999% puratonic salts), CdCl2 (Alfa aesar 99.999% puratonic salts), InCl3 (Alfa aesar 99.999% puratonic salts), EuCl3 (Alfa aesar 99.999% puratonic salts), CoCl2 (Alfa aesar 99.999% puratonic salts), BaCl2(Alfa aesar 99.999% puratonic salts), ZnCl2(Alfa aesar 99.999% puratonic salts), HgCl2 (Alfa aesar 99.999% puratonic salts), CeCl3 (Alfa aesar 99.999% puratonic salts), SmCl3(Alfa aesar 99.999% puratonic salts), YbCl3(Alfa aesar 99.999% puratonic salts). All prepared metal ion, buffer, and gel, and desalting solutions used Milli-Q water with no additional treatment. The pH of relevant solutions was confirmed using the Fisher Scientific Accumet AB15 pH meter. Primers and template DNA were desalted using Waters Sep-Pak columns.

[0123] Sequences: TABLE 2 shows DNA sequences for the in vitro selection of a K+- selective DNAzyme. TABLE 2. DNA sequences.

[0124] In vitro selection: Selection was completed using a 110-mer oligonucleotide with a 50-nt random sequence flanked by defined sequences that can form double-stranded binding arms surrounding rG as the putative cleavage site and then conserved PCR primer-binding sequences at both ends. The full N50 pool was ordered from IDT and used directly after ^Attorney Docket No.10046-637WO1 denaturing PAGE (10% Acrylamide, 8 M urea, 90 mM Tris, 90 mM boric acid, 2.75 mM Na2EDTA) purification. DNAs were extracted in the Li+extraction buffer (0.3 M LiCl, 0.001 M Li2EDTA, 0.01 M Bis-Tris pH 7.0) and standard desalting using a Sep-pak column.

[0125] The partial counter sequence of the N50 pool was ordered and the pool was generated via asymmetric PCR, due to IDT synthesis limitations. To generate the full-length N50 pool for selection, 1x Li+PCR buffer, 6 U / 100 ^L Taq Polymerase, 0.2 mM of each dNTP, 1 ^M P3-rG, 100 nM IDT Template, 1 ^L32P-labeled ^-ATP were mixed and underwent the follow temperature treatment: 3 min at 95°C, repeated 14 x (30 s at 95°C, 1.25 min 60°C, 1.25 min 72°C), 10 min at 72°C, then cooled to 4°C. The 3 mL of DNA product was then desalted using ethanol precipitation in ~70 % EtOH, 10 mM Tris-acetate (Tris-OAc) pH 9.0 chilled at - 80°C for at least 2 hours. Chilled samples then underwent centrifugation at -10°C at 16000 rpm for 30 min then washed with 70 % EtOH and spun down for an additional 20 min at -10°C at 16000 rpm. Sample were then air dried. Dried samples were resuspended in Millipore water and Stop Solution (9.2 M Urea, 2x TB) and purified on a 10 % Acrylamide denaturing PAGE gel. The purified sample was excised from the gel, the extricated gel was crushed, and the sample DNA was extracted in the Li+extraction buffer (0.3 M LiCl, 0.001 M Li2EDTA, 0.01 M Bis-Tris pH 7.0) using a quick extraction technique. Extracted DNA was desalted by adding the Li+precipitation solution 1M Li-acetate (LiOAc) with 1 / 10 volume and ethanol with ~ 2.7 volume, and chilled at -80°C for 2 hours or longer. Samples were spun down at -10°C at 16000 rpm for 30 min. The supernatant was removed, and the sample was washed with chilled (- 20°C) 70 % EtOH for an additional 20 min at -10°C at 16000 rpm. Samples were then air dried.

[0126] For positive selection, samples were resuspended in Milli-Q water then the reaction was initiated by the addition of 2x positive selection buffer (0.3 M KCl, 0.001 M EDTA, Tris pH 7.4). Samples were incubated at room temperature for 2 hours before the addition of 2x Stop Solution. Samples were purified using denaturing PAGE, from which the cleaved sequence was excised. Specific size marker with the same length as the cleaved pool was used to indicate the mobility of cleaved pool for size-selective gel excision and extraction. The pool was extracted using the Li+extraction buffer and desalted using precipitation with LiOAc as introduced above. Pool was subject to PCR based pool regeneration.

[0127] Subsequently, the cleaved DNAzyme were then regenerated by PCR amplification for the next selection round. To regenerate the cleaved N50 pool there is a two-step PCR process. PCR 1 adds extends the cleaved DNA sequence with P2. Subsequently PCR 2 fully restores the extended sequence to the initial full-length DNA pool sequence and reincorporates the RNA active site. PCR 1 used 1x Li+PCR buffer, 6 U / 100 ^L Taq Polymerase, 0.2 mM each ^Attorney Docket No.10046-637WO1 dNTP, 0.6 ^M P1-iSp3, 1 ^M P2, 50 ^L of the 150 ^L of redissolved desalted Positive Selection product, 1 ^L32P-labeled ^-ATP. The PCR 1 mixture underwent the following heat treatment: 3 min at 95°C, repeated 14-22 x (30 s at 95°C, 1.25 min 60°C, 1.25 min 72°C), 10 min at 72°C, then the mixture is cooled to 4°C. PCR 2 used 1x Li+PCR buffer, 6U Taq, 0.2 mM each dNTP, 1 ^M P1-iSp3, 1 ^M P3-rA, 5 ^L of PCR 1 product, 1 ^L32P-labeled ^-ATP were mixed and underwent the follow temperature treatment: 3 min at 95°C, repeated 14-18 x (30 s at 95°C, 1.25 min 52°C, 1.25 min 72°C), 10 min at 72°C, then cooled to 4°C. The remaining PCR 1 is purified by PCR purification kit (QIAGEN) and stored at -20°C. All of the PCR 2 product is PAGE purified. The full-length pool region was excised from the gel. The regenerated pool was extracted by the Li+extraction buffer and was desalted using ethanol precipitation with LiOAc as introduced above.

[0128] The negative selection was not introduced until a significant amount of pool was cleaved from the previous round. After 6 rounds of positive selection, the selection pool started to show detectable cleaved products after 2 hours of incubation in the selection buffer. Thus, the negative selection is introduced post the round 6 of the K+-DNAzyme selection right after the PCR pool regeneration step and before the positive selection step.

[0129] For negative selection, samples were resuspended in Milli-Q water then the reaction was initiated by the addition of 2x negative selection buffer (0.3 M NaCl or LiCl, 0.001 M EDTA, Tris pH 7.4). To keep the ionic strength consistent between selection steps, negative selections were conducted using either 150 mM LiCl or 150 mM NaCl by alternating the negative selection target metal ions for each iterative cycle. Samples were incubated at room temperature for 2 hours before the addition of 2x Stop Solution. Samples were purified using denaturing PAGE, from which the full-length non-cleaved sequence was excised. Specific size marker with the same length as the cleaved pool was used to indicate the mobility of cleaved pool for size-selective gel excision and extraction. The pool was extracted using the Li+extraction buffer and desalted using precipitation with LiOAc as introduced above. Pool was subject to the positive selection directly.

[0130] After a total of 10 rounds of selections, the activity of the pool plateaued and did not show further increase. Sequencing library was prepared from the PCR product from round 10 selection pool using the Nextera XT DNA Library Preparation Kit and purified with AMPure XP beads. The library was quality controlled by Agilent 2100 Bioanalyzer and quantified by Qubit dsDNA Quantification Assay Kits before NGS in the DNA Services Lab at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana Champaign. ^Attorney Docket No.10046-637WO1

[0131] To identify the conserved catalytic core sequence responsible for the K+-dependent activity, the study used OligoAnalyzer to predict potential secondary structures of the above DNAzyme sequence in 150 mM monovalent metal ion and 2 mM Mg2+at 37°C.

[0132] PAGE based Activity Assay: Activity assays were carried out to determine the best round of selection and selection conditions to submit for sequencing, to characterize individual sequences. Activity assay experiments were carried out with incorporating either32P-labeled or FAM fluorophore labeled fP2-rG DNA into full-length selection pool or candidate sequences. The labeled full-length DNAs were PCR generated, PAGE purified, and dissolved in Milli-Q H2O for activity assays. DNA was mixed with 2x reaction buffer to initiate the reaction. Selectivity of the sensor for K+over other metal ions was tested in the presence of 13 different metal salts. Sensor response to monovalent competing metal ions (Li+, Na+, Rb+, Cs+) was tested at 100 mM, while divalent (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Cu2+, Fe2+, Co2+, Ni2+) and trivalent (Fe3+) metal ions were tested at 2 mM and 0.2 mM, respectively.

[0133] At predetermined timepoints 2 ^L aliquots of the reaction mixture were removed and added to 20 ^L of Stop Solution with loading dyes (9.2 M Urea, 0.001 M Li2EDTA, 180 mM Tris, 180 mM boric acid, 0.05 % xylene cyanol, 0.05 % bromophenol blue). Samples were resolved by 10 % denaturing PAGE gels.32P gels were wrapped in cellophane and exposed to a phosphoimage film, the image was captured using a Molecular Dynamics Storm 430 Phosphorimager (from Amersham Biosciences). Fluorescence gels were directly imaged under the Gel Doc XR+Gel Documentation System (Biorad). The total percent of DNA cleaved at each timepoint was quantified using the Image Quant Software (Molecular Dynamics) or the Image Lab Software (v6.1, Biorad). Each image was adjusted for background and quantified for bands intensity. The reaction rate for the candidate DNAzyme sequences was obtained using the equation below, in which t is time, [P]t is the cleavage at time t, [P]^ is the final cleavage ratio, kobs is the pseudo-first order rate constant.

[0134] In vitro Fluorescence Activity Assay: The enzyme–substrate complex was formed by annealing a mixture of 20-4 enzyme and 20-4 substrate strands for sensor in buffer C with the ratio of 1.1:1. Concentrated Li+or other metal solutions in buffer C were mixed with annealed complex before recording the fluorescence change. The final concentration of the sensor complex was calculated to be 50 nM. The fluorescence change was continuously monitored by a FluoroMax-2 fluorometer (Horiba Jobin Yvon, Inc., Edison, NJ) every 30 min ^Attorney Docket No.10046-637WO1 for at least 6 h. The excitation wavelength was 488 nm, and the emission was monitored at 520 nm.

[0135] Breast Tissue Cell Line Culture and Transfection: MCF-7, MCF-10A, and MDA cell line was directly purchased from ATCC and cultured in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin, in 25 cm2culture flasks at 37°C in a humidified 5% CO2incubator. Before imaging, cells were plated in 35 mm glass-bottom dishes (MatTek, Cat: P35GC-1.0-14-C) or glass-bottom eight-well chamber slides (ibidi, Cat. No.80806) and grown to 50–70% confluence.

[0136] The DNAzyme sensor was added into the cell culture Petri dish together with PEI transfection reagent (Sigma-Aldrich). The working DNAzyme sensor concentration is estimated to be 200 nM final concentration. The working DNAzyme sensor concentration is estimated to be 800 nM final concentration. After 4 h transfection, cells were washed thoroughly with PBS to remove the excess amount of DNAzyme in the medium.

[0137] Intracellular K+Imaging: After transfection and washing steps with DPBS, breast tissue or immune cells were imaged under either Zeiss LSM 710 NLO or Zeiss LSM 880 confocal microscope at 20× and 63× magnification. Fluorescence emission of Hoechst 33258 was measured over 415–475 nm ranges, with excitation at 405 nm. Fluorescence emission of LysoTracker was obtained by exciting at 561 nm and measuring over 570–735 nm. Fluorescence emission of FAM or Alexa488 was obtained by exciting at 488 nm and measuring over 497–550 nm. The pinhole and gain settings were kept constant throughout the whole imaging process. Results

[0138] In vitro Selection of K+-Specific RNA-cleaving DNAzymes: To obtain K+-selective RNA-cleaving DNAzymes, the study conducted in vitro selection using a previously reported protocol with modifications as described in the Method section. Specifically, the study started with a DNA library containing 50 randomized sequences and performed positive selection in 150 mM K+in Tris buffer at pH 7.4 and 37°C to mimic the intracellular K+concentration and conditions. The DNAzymes capable of catalyzing rG cleavage in the presence of K+was isolated by denaturing polyacrylamide gel electrophoresis (PAGE), which were then amplified by polymer chain reaction (PCR) and subjected to the next round of selection. To enhance the selectivity of the DNAzymes for K+against other metal ions, starting in Round 7, the study added a counter selection step prior to the positive selection step by incubating the DNA pools with the same buffer containing Li+and Na+and removing the DNA populations that would ^Attorney Docket No.10046-637WO1 cleave in the presence of these other metal ions. To increase the stringency of the counter selections, the study extended the incubation time with the other metal ions from 2 hours in Round 7 to 16 hours in Round 10 and sequenced the Round 10 selection pool.

[0139] After analyzing the sequences to identify the top representing sequences, the study performed activity assays and found one sequence with the highest selectivity for K+over Na+for further study. The predicted Structure 9 returned by OligoAnalyzer matches what was designed, e.g., two binding arms flanking the rG cleaving site and is named as “cis-10-9 DNAzyme” (FIG. 1A). To make the DNAzyme more effective for sensing applications, the redundant sequences of cis-10-9 DNAzyme nucleotides was truncated, and the resulting truncated DNAzyme was further converted into an enzyme and a substrate strands (called 10- 9 DNAzyme hereafter, see FIG.1B). Using PAGE-based activity assay, it was found that the trans-10-9 DNAzyme displayed a physiologically relevant K+response range from 10 mM to 250 mM of K+(FIG. 2A). A plot of the observed rates of ribonucleotide site cleavage vs. concentrations of K+indicates an apparent dissociation constant (Kd) of ~ 105 mM (FIG.2B). The 10-9 DNAzyme is the first K+specific RNA-cleaving catalytic nucleic acids with a high selectivity.

[0140] DNAzyme-Based Fluorescent Sensor for K+Detection: To convert the K+- dependent catalytic activity of the 10-9 DNAzyme into a turn-on fluorescence sensor, the study next designed a sensor based on the “catalytic beacon” approach by labeling the substrate strand of the 10-9 DNAzyme with a 6-carboxyfluorescein fluorophore (FAM) at its 5’ end and the enzyme strand of the 10-9 DNAzyme with an Iowa Black FQ quencher at its 3’ end. In addition, a second quencher was added at the 3’ end of the substrate strand to minimize background fluorescence due to the possibility that a small portion of the substrate strand is dehybridized from the enzyme strand in the absence of DNAzyme-based cleavage (FIG.3A).

[0141] The sensor displayed an increasing fluorescence signal with increasing concentrations of K+(FIG. 3B), including a linear response up to 200 mM (FIG. 3C) with determined limit of detection to be 23.21 mM (3^ / slope), suggesting that this sensor is useful for quantitative K+imaging under cellular K+levels. To rule out any artifacts due to nonspecific DNA cleavage, the study introduced a mutation in the middle of a predicted stem structure of the K+DNAzyme to destabilize the DNA hybridization and change the overall predicted structure of the DNAzyme (FIG. 3D). This single-mutation was enough to render the 10-9 DNAzyme completely inactive, as no elevated fluorescence signals were detected in the presence of K+when using this inactive DNAzyme as the negative control (FIG. 3E). It was ^Attorney Docket No.10046-637WO1 found that the sensor exhibited >1000-fold selectivity for K+over Na+and > 100-fold selectivity over other major physiological relevant metal ions (FIG.3F).

[0142] Intracellular K+Imaging Using the 10-9 K+DNAzyme: To investigate the ability of this 10-9 K+DNAzyme sensor to detect intracellular K+in living cells, the study chose the HeLa CCL-2 adenocarcinoma cell line, a widely used model for living cell sensor development, as a proof-of-concept cell model for K+sensor optimization and validation. The sensor showed significant fluorescence turn-on after its delivery, indicating that the K+DNAzyme sensor effectively detected intracellular K+levels (FIG. 4A). Moreover, higher signal was observed in nuclei from K+DNAzyme imaging, indicating nuclear enrichment of K+, which is consistent with previous research on higher K+levels in cell nuclei . In contrast, much lower fluorescence signal was observed from the inactive DNAzyme sensor negative control, supporting that the signals from cell imaging reflect mostly the K+concentration in cells (FIG.4B).

[0143] To validate the capability of the 10-9 K+DNAzyme sensor in differentiating intracellular K+level changes, the study employed known ionophores to up- or down-regulate the cellular K+levels and recorded the signal levels from the DNAzyme sensor. Specifically, when 10 µM valinomycin, which is a highly selective lipophilic K+ionophore that would act as a shuttle for K+, was added to balance the intracellular K+levels with extracellular media levels of K+, a decrease of the fluorescent signal was observed, due to K+efflux from cell body to media. When 75 and 150 mM K+were further added in the cellular media, an increase of the fluorescent signal was observed (FIG. 5A). Statistical data quantifying the fluorescence intensity showed that the cellular signal intensity from K+sensor positively correlates with the extracellular media concentrations of K+(FIG.5B).

[0144] To further validate cellular K+regulation results, the study added different concentrations of 18-crown-6, a crown ether that bound K+in the cells media to downregulate K+availability to cells and observed decreased fluorescent signals with increasing levels of K+chelator (FIGS.5C-5D). Together, these results demonstrated the ability of using the 10-9 K+DNAzyme sensor in live imaging of different cellular K+levels.

[0145] Decreases in K+Levels in Breast Tissue Cells During Cancer Metastasis: To demonstrate the ability of the DNAzyme sensor in studying K+level changes across different metastatic stages, the study delivered the sensor using a PEI-based transfection method to MCF-10A, MCF-7, and MDA-MB-231, which are commonly used human breast cell lines to mimic benign, non-invasive cancerous, and metastatic cancer states, respectively (FIG.6A).

[0146] To account for any artifacts, the study also used the same method to deliver the inactive DNAzyme into the same set of cell lines and used the signal from this inactive sensors ^Attorney Docket No.10046-637WO1 (iE) (FIG. 7) as a reference to calculate the ratio of fluorescence signal intensity from active over inactive sensors for each cell type. The results shown in FIG. 6B revealed high endogenous K+levels in normal MCF-10A cells, less K+level in non-invasive MCF-7 cells and the lowest K+level in the invasive MDA-MB-231 cells, suggesting that invasive cancer cells may contain a reduced intracellular K+levels.

[0147] High environmental K+levels contribute to resistance to anti-cancer drugs: The cancer microenvironment is often characterized by abnormally high K+levels, which impair immune functions and aid cancer cell survival. It is intriguing to consider whether cancer cells exploit K+-rich environments to disrupt chemical cancer therapies. The study explored the effects of high environmental K+levels on tumor survival by evaluating the K+channel- dependent anticancer molecule ML133 on MDA-MB-231 cells cultured in high and low K+conditions.

[0148] Given the above findings of different K+levels in different types of cancer cells, the study attempted to use the DNAzyme sensors to monitor K+levels in cancer cells when they are treated with anti-cancer drugs and to gain insights into the relationship between the K+levels and drug effect. To achieve this goal, MDA-MB-231 cells were stained with either SYTO 82, a cell-permeant orange fluorescent nucleic acid stain, for total cell quantification, or with SYTOX Green, a cell-impermeant dye, to stain dead cells with a compromised membrane integrity. As shown in FIG. 8A, when the cells were treated with ML133, a K+channel- dependent anticancer drug, fewer cell death was observed when 40 mM of K+was added to the cell media. This effect was confirmed by MTT assays (FIG.8B), indicating that the anticancer drug efficacy of ML133 is hampered by high K+levels. ML133 is known to inhibit the Kir2.1 K+channel, blocking inward K+flow. Consistent with this function, decreased K+accumulation was observed with increased ML133 concentration (FIG. 8C). Cells in high K+media showed elevated intracellular K+levels and reduced cell death (FIG. 8D), suggesting that high K+in the tumor microenvironment confers resistance to ML133 by compensating for decreased intracellular K+levels.

[0149] To corroborate the above finding, the study further tested if this drug resistance applies to other anticancer drugs affecting membrane potentials, given that environmental ion concentration influences these potentials. The study evaluated Amiodarone, an antiarrhythmic and anticancer drug that induces plasma membrane hyperpolarization. High K+levels in culture media attenuated the effectiveness of Amiodarone in inhibiting MDA-MB-231 cell growth (FIGS. 9A-9B). Treatment with low (5 ^M) and high (above 15 ^M) Amiodarone concentrations led to slight increases and significant decreases in intracellular K+levels, ^Attorney Docket No.10046-637WO1 respectively (FIG. 9C). High environmental K+was able to compensate for intracellular K+decreases and rescue cell viability (FIG. 9D), suggesting that high K+levels in the tumor microenvironment confer drug resistance across multiple anticancer drugs. Discussion

[0150] This study developed a K+-specific DNAzyme with high selectivity over competing metal ions, including Na+, commonly found in biological systems. Most commercial K+sensors do not achieve this level of selectivity. This selectivity, combined with inactive DNAzyme negative control, ensures that signals reflect K+concentrations rather than artifacts from other ions, nucleases, or strand dehybridization. This sensor also has a broad detection range up to 200 mM K+for cellular applications, which is not usually achievable by genetically encoded or DNA based K+sensors. Using these advantages, the study created a catalytic beacon fluorescent sensor for live cell K+imaging across various cell types, including breast cancer cells at different metastatic stages and monocyte cell lines at various differentiation stages.

[0151] It is intriguing to connect K+level changes during cancer metastasis with anticancer drug resistance and high microenvironmental K+levels. The elevated K+levels in cancer microenvironment impair T-cell function by accelerating senescence. Understanding how cancer cells create K+-rich microenvironments to interfere with chemotherapy is crucial. K+imaging revealed that breast cancer cells maintain lower intracellular K+levels than cell lines from the same tissue, with the lowest levels in metastatic cells. Despite MDA-MB-231 cells being cultured in higher K+media than MCF-10A cells, they have lower intracellular K+concentrations. This suggests metastatic cells may actively export K+during progression, contributing to high microenvironmental K+. It is unclear if decreased cellular K+serves as an extracellular resource buffering against chemotherapy. The K+sensor provides insights into the link between cancer cells, their microenvironment, and drug resistance. Future studies on K+transport and function could elucidate the function of K+in tumor microenvironments and drug resistance, aiding cancer treatment development. Conclusion

[0152] This study developed a K+-specific DNAzyme with high selectivity for K+over Na+and other biologically relevant metal ions. This DNAzyme exhibits a detection range suitable for intracellular K+measurements. The study used it to develop a catalytic beacon fluorescent sensor for intracellular K+imaging in various cell lines, including human breast tissue cells. Notably, the sensor revealed different K+accumulation patterns in normal, non-invasive, and invasive breast tissue cells. Coupled with observations of altered K+levels under anticancer drug treatment in high and normal K+media, these findings suggest a K+regulation system in ^Attorney Docket No.10046-637WO1 cancer tissues that creates a K+-rich microenvironment conducive to cancer cell survival during drug treatment. These insights demonstrate that the K+sensor holds potential as a powerful tool for investigating K+dynamics in biological systems and advancing understanding of K+in cancer biology. Example 2: Decoding Potassium Homeostasis in Cancer Metastasis and Drug Resistance: Insights from a Highly Selective DNAzyme-Based Intracellular K+Sensor

[0153] Potassium ions (K+) are integral to a wide range of physiological processes, including electrochemical regulation, cellular homeostasis, and metabolic signaling [1]. As the most abundant intracellular cation, K+plays a fundamental role in maintaining membrane potential, which is crucial for neuronal excitability, muscle contraction, and cardiac rhythm [2,3]. It also serves as a key regulator of cellular homeostasis, ensuring proper cell volume control, pH balance, and ionic equilibrium [4,5]. Beyond these roles, K+is involved in metabolic and signaling pathways, including enzyme activation and cell fate decisions such as apoptosis and proliferation [6–8]. One particularly intriguing context for K+research is the tumor microenvironment, where aberrant ion homeostasis has been implicated in disease progression. The observation of elevated extracellular K+concentrations within the tumor microenvironment raises important questions about their role in cancer progression and treatment resistance [9,10]. Recent research has demonstrated that high K+levels in the tumor microenvironment can suppress T-cell activity, creating an immunosuppressive milieu that benefits cancer cells [10–14]. Understanding whether cancer cells actively maintain high K+levels in their environment to gain survival advantages is crucial. Exploring the relationship between K+and cancer drug resistance would be particularly impactful in uncovering novel mechanisms underlying cancer persistence and recurrence. Such discoveries, while significant for cancer research, also underscore the broader relevance of understanding K+dynamics in health and disease. Given the multifaceted role of K+in cell apoptosis, immunology, and cancer biology, developing chemical biology tools to accurately probe K+levels is of great significance [15,16]. These tools will provide a platform for exploring K+biology in diverse systems by enabling visualization and cross-comparison of K+homeostasis across cancer cell lines with varying metastatic stages and metabolic profiles. Insights gained from such tools could lead to novel therapeutic strategies aimed at manipulating K+concentrations or K+channel activities to enhance the effectiveness of cancer treatments and potentially overcome certain types of drug resistance.

[0154] With these significant impacts on many areas of biochemical and biomedical science, intensive research has been performed on developing K+imaging in living cells. ^Attorney Docket No.10046-637WO1 Among these sensory probes, small molecule-based imaging probes are the most broadly studied [17–19]. However, current commercially available small molecular K+sensors share a major limitation of low selectivity of K+over other biorelevant metal ions, especially Na+. To overcome this limitation, new small molecular and small molecule-containing nanoparticle sensors for K+have been reported, but they exhibit good performance mainly in a narrow pH range and many display small relative signal changes. The majority of these sensors that function under physiological conditions necessitate advanced chemical and nanomaterial syntheses [20,21], making them less readily accessible to researchers. As an alternative to the small molecular sensors, genetically encoded indicators based on naturally existing Escherichia coli K+binding protein (Kbp) have been successfully developed for selective K+detection. The early versions of the genetically encoded K+sensors (KIRIN1, KIRIN1-GR, GINKO1, GEPIIs) have Kd or EC50 less than 1 mM for K+binding, which allows for imaging of K+at micromolar and low millimolar concentration range [22–26]. Further screening of the genetically encoded K+sensors identified a series of mutants for K+imaging at a moderately higher concentration range, with the GINKO2 sensor showing Kd at 15.3 mM and lc-LysM GEPII 1.0 showing EC50 at 27.43 mM as the current best versions and have been tested in intracellular K+imaging. Given that the average cellular level of K+is between 140-150 mM [27–29], it is desirable to develop K+sensors that can function well in this concentration range. DNA-based sensors have intrinsic advantages in biocompatibility, are easier to synthesize over small molecular sensors, and have higher stability and smaller size compared with protein sensors. Given the advantages, a sensor that conjugates a small molecular K+sensor with DNA has been reported

[0019] , but this sensor shares similar limitations to small molecular sensors, such as difficult synthesis, moderate selectivity for K+over Na+and other biorelevant metal ions, and a Kd misaligned with the high intracellular K+concentration typically encountered in research. On the other hand, a few DNA-based K+sensors have been reported taking advantage of DNA sequences that may fold into G-rich quadruplex or triplex structures in the presence of K+(e.g., the thrombin binding aptamers) [30–32]. However, this type of K+sensors share the same limitations of suboptimal detection range confined to the micromolar to low millimolar window, high background in detection, and inconsistent performance arising from variations in G-quadruplex topology, which is sensitive to ionic strength and the presence of competing ions.

[0155] To overcome the limitations of current K+sensors, a study was conducted to develop DNAzyme-based sensors. First discovered in 1994, DNAzymes are a class of DNA molecules that display enzymatic activities in the presence of a metal ions as a cofactor

[0033] . ^Attorney Docket No.10046-637WO1 Among DNAzymes, RNA-cleaving DNAzymes are of particular interest for metal ion sensing due to their fast reaction rate, high metal ion cofactor selectivity, and ease of converting the cleavage into a detectable signal by the catalytic beacon strategy [34,35]. Through a combinatorial strategy named “in vitro selection”, this study can isolate DNAzymes that are highly selective by introducing counter selection against competing metal ions, that are fast and active by adjusting the incubation time of positive selection, and most importantly, that are suitable to certain concentration dynamic range by tuning target metal ion concentrations in positive and counter selections [36–40]. Using this strategy, many metal ion-specific DNAzymes have been selected and turned into fluorescent sensors [41–46], including Zn2+[47–50], Pb2+[51,52], UO22+

[0053] , Mg2+[54,55], Ca2+

[0056] , Cu2+[57,58], Hg2+

[0059] , Ag+[60,61], Na+[62,63], Li+[64,65], Cr3+

[0066] , Fe2+ / 3+

[0067] and lanthanides [68–70], showcasing the generalizability of this strategy in generating highly selective and tunable metal ions sensors. Many of these sensors have been further engineered for imaging metal ions in living cells and organisms [71–73]. However, no K+selective DNAzyme with substantial activity has been reported.

[0156] This study describes in vitro selection and characterization of an RNA-cleaving DNAzyme with high selectivity (> 1000-fold) for K+over other competing ions, with a broad linear detection range up to 200 mM and an estimated Kdof around 105 mM, covering the average cellular K+level of approximately 150 mM. This K+-specific DNAzyme was transformed into a biocompatible fluorescent sensor capable of detecting intracellular K+levels in living cells. The study established this sensing system for visualizing K+homeostasis across human breast cancer cell lines at different cancer stages to determine differential K+accumulation during metastasis. These findings revealed lower K+accumulation in metastatic breast cancer cells compared to normal states and demonstrated that high environmental K+attenuates anti-cancer drug efficiencies through compensating the decreases of intracellular levels of K+during anti-cancer treatment. By developing a highly selective DNAzyme sensor that can image K+in the physiologically relevant levels of K+in living cells and gaining deeper insights into how cells response to K+in the tumor microenvironment, this work opens new avenues for selectively detecting K+in many biological systems and provides opportunities to further study the K+biochemistry in cancer biology and tumor microenvironments. Methods and Materials

[0157] Materials: All DNA sequences were purchased from Integrated DNA Technologies (IDT) with standard desalting. Prior to use each sequence was purified via denaturing PAGE. Acrylamide / bisacrylamide 40 % solution (29:1) was obtained from Bio-Rad Laboratories, Inc. ^Attorney Docket No.10046-637WO1 The following enzymes, reaction buffers, and reagents used were purchased from New England Biolabs: Taq polymerase, standard Taq buffer, T4-polynucleotide kinase, polynucleotide kinase buffer, and deoxynucleotide (dNTP) solution mix. Both32P labeled ^-ATP and ^-ATP were obtained from Perkin-Elmer. All PCR and32P-labeling experiments were carried out in the BioRad thermocycler.

[0158] The following chemicals were as obtained as follows: 3-(N- morpholino)propanesulfonic acid (MOPS) (Amersham International plc), Urea (Affymetrix, MB grade), Tris (Affymetrix, MB grade), boric acid (Fischer Scientific, electrophoresis grade), Ethylenediaminetetraacetic acid (EDTA) (Fluka, 99.0%), EDTA^2Na^2H2O (Fisher Scientific), 200 proof ethanol (Decon Laboratories, Inc.), sodium acetate, , HCl (Alfa-Aesar, ultrapure), lithium hydroxide (Alfa aesar 99.999% puratonic salts), potassium hydroxide, sodium hydroxide, HCl (Alfa-Aesar, ultrapure). All of the metal salts used were obtained as listed: LiCl (Alfa aesar 99.999% puratonic salts), NaCl (Alfa aesar 99.999% puratonic salts), KCl (Alfa aesar 99.999% puratonic salts), CsCl (Alfa aesar 99.999% puratonic salts), RbCl (Alfa aesar 99.999% puratonic salts), MgCl2 (Alfa aesar 99.999% puratonic salts), CaCl2 (Alfa aesar 99.999% puratonic salts), PbCl2 (Alfa aesar 99.999% puratonic salts), MnCl2(Alfa aesar 99.999% puratonic salts), NiCl2 (Alfa aesar 99.999% puratonic salts), CdCl2 (Alfa aesar 99.999% puratonic salts), CoCl2(Alfa aesar 99.999% puratonic salts), ZnCl2(Alfa aesar 99.999% puratonic salts). All prepared metal ion, buffer, gel, and desalting solutions used Milli-Q water with no additional treatment. The pH of relevant solutions was confirmed using the Fisher Scientific Accumet AB15 pH meter. Primers and template DNA were desalted using Waters Sep-Pak columns.

[0159] In vitro selection: Selection was completed using a 110-mer oligonucleotide with a 50-nt random sequence flanked by defined sequences that can form double-stranded binding arms surrounding rG as the putative cleavage site and then conserved PCR primer-binding sequences at both ends. The full N50 pool was ordered from IDT and used directly after denaturing PAGE (10% Acrylamide, 8 M urea, 90 mM Tris, 90 mM boric acid, 2.75 mM Na2EDTA) purification. DNAs were extracted in the Li+extraction buffer (0.3 M LiCl, 0.001 M Li2EDTA, 0.01 M Bis-Tris pH 7.0) and standard desalting using a Sep-pak column.

[0160] The partial counter sequence of the N50 pool was ordered and the pool was generated via asymmetric PCR, due to IDT synthesis limitations. To generate the full-length N50 pool for selection, 1x Li+PCR buffer, 6 U / 100 ^L Taq Polymerase, 0.2 mM of each dNTP, 1 ^M fP2-rG, 100 nM IDT Template, 1 ^L32P-labeled ^-ATP were mixed and underwent the follow temperature treatment: 3 min at 95°C, repeated 14 x (30 s at 95°C, 1.25 min 60°C, 1.25 ^Attorney Docket No.10046-637WO1 min 72°C), 10 min at 72°C, then cooled to 4°C. The 3 mL of DNA product was then desalted using ethanol precipitation in ~70 % EtOH, 10 mM Tris-acetate (Tris-OAc) pH 9.0 chilled at - 80°C for at least 2 hours. Chilled samples then underwent centrifugation at -10°C at 16000 rpm for 30 min then washed with 70 % EtOH and spun down for an additional 20 min at -10°C at 16000 rpm. Sample were then air dried. Dried samples were resuspended in Millipore water and Stop Solution (9.2 M Urea, 2x TB) and purified on a 10 % Acrylamide denaturing PAGE gel. The purified sample was excised from the gel, the extricated gel was crushed, and the sample DNA was extracted in the Li+extraction buffer (0.3 M LiCl, 0.001 M Li2EDTA, 0.01 M Bis-Tris pH 7.0) using a quick extraction technique. Extracted DNA was desalted by adding the Li+precipitation solution 1M Li-acetate (LiOAc) with 1 / 10 volume and ethanol with ~ 2.7 volume, and chilled at -80°C for 2 hours or longer. The samples were spun down at -10°C at 16000 rpm for 30 min. The supernatant was removed, and the sample was washed with chilled (-20°C) 70 % EtOH for an additional 20 min at -10°C at 16000 rpm. The samples were then air dried.

[0161] For positive selection, samples were resuspended in Milli-Q water then the reaction was initiated by the addition of 2x positive selection buffer (0.3 M KCl, 0.001 M EDTA, Tris pH 7.4). Samples were incubated at room temperature for 2 hours before the addition of 2x Stop Solution. Samples were purified using denaturing PAGE, from which the cleaved sequence was excised. A specific size marker with the same length as the cleaved pool was used to indicate the mobility of cleaved pool for size-selective gel excision and extraction. The pool was extracted using the Li+extraction buffer and desalted using precipitation with LiOAc as introduced above. The pool was subject to PCR based pool regeneration.

[0162] Subsequently, the cleaved DNAzyme were then regenerated by PCR amplification for the next selection round. To regenerate the cleaved N50 pool there is a two-step PCR process. PCR 1 adds extends the cleaved DNA sequence with P2. Subsequently PCR 2 fully restores the extended sequence to the initial full-length DNA pool sequence and reincorporates the RNA active site. PCR 1 used 1x Li+PCR buffer, 6 U / 100 ^L Taq Polymerase, 0.2 mM each dNTP, 0.6 ^M rP1-iSp, 1 ^M fP1, 50 ^L of the 150 ^L of redissolved desalted Positive Selection product, 1 ^L32P-labeled ^-ATP. The PCR 1 mixture underwent the following heat treatment: 3 min at 95°C, repeated 14-22 x (30 s at 95°C, 1.25 min 60°C, 1.25 min 72°C), 10 min at 72°C, then the mixture is cooled to 4°C. PCR 2 used 1x Li+PCR buffer, 6U Taq, 0.2 mM each dNTP, 1 ^M rP1-iSp, 1 ^M fP2-rG, 5 ^L of PCR 1 product, 1 ^L32P-labeled ^-ATP were mixed and underwent the follow temperature treatment: 3 min at 95°C, repeated 14-18 x (30 s at 95°C, 1.25 min 52°C, 1.25 min 72°C), 10 min at 72°C, then cooled to 4°C. The ^Attorney Docket No.10046-637WO1 remaining PCR 1 is purified by PCR purification kit (QIAGEN) and stored at -20°C. All of the PCR 2 products are PAGE purified. The full-length pool region was excised from the gel. The regenerated pool was extracted by the Li+extraction buffer and was desalted using ethanol precipitation with LiOAc as introduced above.

[0163] The counter selection was not introduced until a significant amount of pool was cleaved from the previous round. After 6 rounds of positive selection, the selection pool started to show detectable cleaved products after 2 hours of incubation in the selection buffer. Thus, the counter selection is introduced post the round 6 of the K+-DNAzyme selection right after the PCR pool regeneration step and before the positive selection step.

[0164] For counter selection, samples were resuspended in Milli-Q water then the reaction was initiated by the addition of 2x counter selection buffer (0.3 M NaCl or LiCl, 0.001 M EDTA, Tris pH 7.4). To keep the ionic strength consistent between selection steps, the counter selections were conducted using either 150 mM LiCl or 150 mM NaCl by alternating the counter selection target metal ions for each iterative cycle. Samples were incubated at room temperature for 2 hours before the addition of 2x Stop Solution. Samples were purified using denaturing PAGE, from which the full-length non-cleaved sequence was excised. A specific size marker with the same length as the cleaved pool was used to indicate the mobility of cleaved pool for size-selective gel excision and extraction. The pool was extracted using the Li+extraction buffer and desalted using precipitation with LiOAc as introduced above. Pool was subject to the positive selection directly.

[0165] After a total of 10 rounds of selections, the activity of the pool plateaued and did not show further increase. The sequencing library was prepared from the PCR product from round 10 selection pool using the Nextera XT DNA Library Preparation Kit and purified with AMPure XP beads. The library was quality controlled by Agilent 2100 Bioanalyzer and quantified by Qubit dsDNA Quantification Assay Kits before NGS in the DNA Services Lab at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana Champaign.

[0166] Paired-end sequencing was performed with the Illumina platform. To identify the candidate of DNAzyme from NGS data, the study generated a robust pipeline for analyzing the sequencing results. Firstly, the study evaluated the sequencing quality with fastqc. The sequences with low sequence quality (score<30) were removed. More than 99% of the sequences showed good quality and remained for the consequential analysis. The pair-ended sequencing results were assembled with PEAR. Since the sequencing library was generated with T4-based ligation, which did not preserve the strand information during the next generation sequencing, the sequences assembled are a mixture of DNA containing both ^Attorney Docket No.10046-637WO1 orientations. To preserve all the sequences and identify the correct orientation of the DNAzyme sequences, all the sequencing results were duplicated by generating their reverse complementary sequences and merging into the original library. Then the sequence orientation was identified with the primer sequence, and the sequence with incorrect orientation was removed with Cutadapt. The low-complexity sequences and primer barcode were removed as well. To reduce sequencing bias, the library was divided into two lanes during sequencing and merged at this step. FASTAptamer was utilized to count the sequences and convert their weight into their copy number in the sequenced library (reads per million, RPM) to evaluate their percental in the library. The top 4000 sequences were clustered based on sequence similarity allowing 7 mutations. The enrichment and evolution of the sequences were evaluated based on their RPM over different rounds of selection. The top candidates were identified as the clusters that showed the highest copy number and largest fold-changes over the rounds of selections. To identify the conserved catalytic core sequence responsible for the K+-dependent activity, the study used OligoAnalyzer to predict potential secondary structures of the above DNAzyme sequence in 150 mM monovalent metal ion and 2 mM Mg2+at 37°C.

[0167] PAGE based Activity Assay: Activity assays were carried out to determine the best round of selection and selection conditions to submit for sequencing, to characterize individual sequences. Activity assay experiments were carried out by incorporating either32P-labeled or FAM fluorophore labeled fP2-rG DNA into full-length selection pool or candidate sequences. The labeled full-length cis-DNAzymes were PCR generated, PAGE purified, and dissolved in Milli-Q H2O for activity assays. The trans-DNAzymes were annealed in Tris pH7.4 buffer with 2 µM substrate and 40 µM enzyme strands (enzyme strand to substrate strand ratio of 20:1). DNA was mixed with 2x metal reaction buffer to initiate the reaction. Selectivity of the sensor for K+over other metal ions was tested in the presence of 13 different metal salts. Sensor response to monovalent competing metal ions (Li+, Na+, Rb+, Cs+) was tested at 100 mM, while divalent (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Cu2+, Fe2+, Co2+, Ni2+) and trivalent (Fe3+) metal ions were tested at 2 mM and 0.2 mM, respectively.

[0168] At predetermined timepoints 2 ^L aliquots of the reaction mixture were removed and added to 20 ^L of Stop Solution with loading dyes (9.2 M Urea, 0.001 M Li2EDTA, 180 mM Tris, 180 mM boric acid, 0.05 % xylene cyanol, 0.05 % bromophenol blue). Samples were resolved by 10 % denaturing PAGE gels.32P gels were wrapped in cellophane and exposed to a phosphoimage film, the image was captured using a Molecular Dynamics Storm 430 Phosphorimager (from Amersham Biosciences). Fluorescence gels were directly imaged under the Gel Doc XR+Gel Documentation System (Biorad). The total percentage of DNA cleaved ^Attorney Docket No.10046-637WO1 at each timepoint was quantified using the Image Quant Software (Molecular Dynamics) or the Image Lab Software (v6.1, Biorad). Each image was adjusted for background and quantified for bands intensity. The reaction rate for the candidate DNAzyme sequences was obtained using the equation below, in which t is time, [P]t is the cleavage at time t, [P] is the final cleavage ratio, kobs is the pseudo-first order rate constant.

[0169] In vitro Fluorescence Activity Assay: The enzyme–substrate complex was formed by annealing a mixture of the 10-9 enzyme and 10-9 substrate strands for sensor in Tris pH7.4 buffer with the ratio of 1.1:1. Concentrated K+or other metal solutions in Tris pH7.4 buffer were mixed with annealed complex before recording the fluorescence change. The final concentration of the sensor complex was calculated to be 50 nM. The fluorescence change was continuously monitored by a FluoroMax-2 fluorometer (Horiba Jobin Yvon, Inc., Edison, NJ) every 30 min for at least 6 h. The excitation wavelength was 488 nm, and the emission was monitored at 520 nm.

[0170] Breast Tissue Cell Line Culture and Transfection

[0171] MCF-7, MCF-10A, and MDA cell lines were directly purchased from ATCC and cultured in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin, in 25 cm2culture flasks at 37°C in a humidified 5% CO2incubator. Before imaging, cells were plated in 35 mm glass-bottom dishes (MatTek, Cat: P35GC-1.0-14-C) or glass-bottom eight-well chamber slides (ibidi, Cat. No.80806) and grown to 50–70% confluence.

[0172] The DNAzyme sensor was added into the cell culture Petri dish together with PEI transfection reagent (Sigma-Aldrich). The working DNAzyme sensor concentration is estimated to be 200 nM final concentration. The working DNAzyme sensor concentration is estimated to be 800 nM final concentration. After 4 h transfection, cells were washed thoroughly with PBS to remove the excess amount of DNAzyme in the medium.

[0173] Intracellular K+Imaging: After transfection and washing steps with DPBS, breast tissue were imaged under either Zeiss LSM 710 NLO or Zeiss LSM 880 confocal microscope at 20× and 63× magnification. Fluorescence emission of Hoechst 33258 was measured over 415–475 nm ranges, with excitation at 405 nm. The fluorescence emission of LysoTracker was obtained by exciting at 561 nm and measuring over 570–735 nm. Fluorescence emission of ^Attorney Docket No.10046-637WO1 FAM or Alexa488 was obtained by excitement at 488 nm and measuring over 497–550 nm. The pinhole and gain settings were kept constant throughout the whole imaging process. Results and Discussion

[0174] In vitro Selection of K+-Specific RNA-Cleaving DNAzymes: To obtain K+-selective RNA-cleaving DNAzymes, the study conducted in vitro selection using a previously reported protocol (see sequences in TABLE 2)

[0064] . Briefly, the study started with a DNA library with 50 positions containing randomized sequences and performed positive selection to isolate DNAzymes capable of catalyzing rG cleavage in the presence of K+using a denaturing polyacrylamide gel electrophoresis (PAGE). The concentration for this positive selection is designed to be 150 mM K+in Tris buffer at pH 7.4 and 37°C to mimic the intracellular K+concentration and conditions. To enhance the selectivity of the DNAzymes for K+against other metal ions, starting in Round 7, the study carried out counter selections by incubating the DNA pools with the same buffer containing Li+and Na+prior to the positive selection step and removing the DNA populations that could cleave in the presence of these other metal ions. To increase the stringency of the counter selections, the study extended the incubation time with the other metal ions from 2 hours in Round 7 to 16 hours in Round 10 and sequenced the Round 10 selection pool.

[0175] The study obtained the sequences of the enriched DNA libraries from Round 10 through next-generation sequencing (NGS) and performed activity assays of the top sequences and found one sequence with the highest selectivity for K+over Na+for further study. The predicted Structure 9 returned by OligoAnalyzer matches what was designed, e.g., two binding arms flanking the rG cleaving site and is named as “cis-10-9 DNAzyme” (FIG. 1A). To facilitate the synthesis of DNAzyme and the design of “catalytic beacon” for fluorescent sensing of K+(vide infra), the study truncated the redundant sequences to produce the trans10- 9 DNAzyme shown in FIG.1B. Using PAGE-based activity assay, it was found that the trans- 10-9 DNAzyme displayed a physiologically relevant K+response range from 10 mM to 250 mM of K+(FIG. 2A). A plot of the observed rates of ribonucleotide site cleavage against concentrations of K+indicates an apparent dissociation constant (Kd) of ~ 105 mM (FIG.2B). The 10-9 DNAzyme is the first K+specific RNA-cleaving catalytic nucleic acid with substantial activity and high selectivity.

[0176] DNAzyme-Based Fluorescent Sensor for K+Detection: To convert the K+- dependent catalytic activity of the 10-9 DNAzyme into a turn-on fluorescence sensor, the study next designed a sensor based on the “catalytic beacon” approach by labeling the substrate strand of the 10-9 DNAzyme with a 6-carboxyfluorescein fluorophore (FAM) at its 5’ end and ^Attorney Docket No.10046-637WO1 the enzyme strand of the 10-9 DNAzyme with an Iowa Black FQ quencher at its 3’ end

[0034] . In addition, a second quencher was added at the 3’ end of the substrate strand to minimize background fluorescence due to the possibility that a small portion of the substrate strand is released from the enzyme strand in the absence of DNAzyme-based cleavage (FIG.10A)

[0053] .

[0177] The sensor displayed an increasing fluorescence signal with increasing concentrations of K+(FIG.10B), including a proximately linear response up to 200 mM (FIG. 10C) with a limit of detection to be 21.10 mM (3^ / slope) (FIG. 10D), suggesting that this sensor is useful for quantitative K+imaging under cellular K+levels. To rule out any artifacts due to nonspecific substrate strand cleavage, the study introduced a mutation in the middle of a predicted stem structure of the K+DNAzyme to destabilize the DNA hybridization and change the overall predicted structure of the DNAzyme (FIG.10E). This single mutation was enough to render the 10-9 DNAzyme completely inactive, as no elevated fluorescence signals were detected in the presence of K+when using this inactive DNAzyme as the negative control (FIG.10F).

[0178] Then, the study evaluated the selectivity of the K+sensor over other metal ions. Different concentrations were used for different metal ions due to the limited solubility of some of the metal ions in aqueous buffers, which made it impractical to apply a uniform concentration across all species. Instead, the study tried to test at the highest concentrations under the physiological conditions for these metal ions. It was found that the K+sensor exhibited >1000-fold selectivity for K+over Na+and > 100-fold selectivity over other major physiological relevant metal ions (FIG.10G). Most commercial K+sensors do not achieve this level of selectivity. This selectivity, combined with inactive DNAzyme negative control, ensures that signals reflect K+concentrations rather than artifacts from other ions, nucleases, or strand dehybridization. This sensor also has a broad detection range up to 200 mM K+for cellular applications, which is not usually achievable by genetically-encoded [22–26], or other DNA-based K+sensors [30–32]. Given these advantages, the study next applied this catalytic beacon fluorescent sensor for live cell K+imaging across various cell types.

[0179] Intracellular K+Imaging Using the 10-9 DNAzyme: To investigate the ability of this novel 10-9 DNAzyme sensor in detecting intracellular K+in living cells, the study chose HeLa CCL-2 adenocarcinoma cell line, a widely used model for living cell sensor development for sensor validation and optimization. The sensor showed significant fluorescence turn-on after its delivery, indicating that the K+DNAzyme sensor effectively detected intracellular K+levels (FIG. 4A). Moreover, a higher signal was observed in nuclei from K+DNAzyme imaging, indicating nuclear enrichment of K+, which is consistent with previous research on ^Attorney Docket No.10046-637WO1 higher K+levels in cell nuclei [74,75]. In contrast, a much lower fluorescence signal was observed from the inactive DNAzyme sensor (iE) as a negative control, supporting that the signals from cell imaging reflect mostly the K+concentration in cells (FIG.11).

[0180] To validate the capability of the 10-9 K+DNAzyme sensor in differentiating intracellular K+level changes, the study employed chemical tools to up- or down-regulate the cellular K+levels and recorded the signal levels from the DNAzyme sensor in response to such regulations. To enhance the efficiency of changing K+levels in cells, the study applied valinomycin, which is a highly selective lipophilic K+ionophore that would insert into the cell membrane and act as a shuttle for K+, to balance the intracellular K+levels with extracellular media levels of K+. An increase in cellular signal change of the sensor when increasing levels of K+is added to the cell media (FIG. 12A). Statistical quantification of the fluorescence intensity showed that the cellular signal intensity from K+sensor positively correlates with the extracellular media concentrations of K+(FIG. 12B). For example, when the HeLa cells were equilibrated with 150 mM K+, the intracellular fluorescent signal was significantly higher than what is observed from the 75 mM extracellular K+group and the untreated group, indicating a cytoplasmic K+level lower than 150 mM.

[0181] To further validate the cellular K+regulation results, the study used a K+chelator, 18-crown-6, to downregulate intracellular K+concentration. The fluorescent signal K+sensor decreased when increasing levels of K+chelator were applied onto cells (FIGS. 12C-12D). These results demonstrated the ability of using the 10-9 DNAzyme sensor for live imaging of cellular K+in different levels.

[0182] K+Homeostasis in Breast Tissue Cells Across Cancer Progression States: To demonstrate the application of the sensors in probing the roles of K+in cancer biology, the study used the sensor to investigate K+concentrations in cell lines representing various stages of cancer progression from the same tissue. The human breast tissue cell lines MCF-10A, MCF-7, and MDA-MB-231 are commonly used models to mimic benign, non-invasive cancerous, and invasive metastatic cancer states, respectively. The K+sensors were introduced into these cell lines to evaluate K+level changes at different stages (FIG. 13A). The study applied a PEI-based transfection method to deliver active and inactive DNAzyme sensors into these cells to avoid artifacts from different transfection reagents. For transfection efficiency and non-specific signal normalization, the study utilized the signal from inactive sensors (iE) as a reference (FIG. 7). The study calculated the fluorescence signal intensity ratio from the active over the inactive sensors for each cell type (FIG. 13B). These results revealed high endogenous K+levels in normal MCF-10A cells, with less endogenous K+levels in non- ^Attorney Docket No.10046-637WO1 invasive MCF-7 cells. The invasive MDA-MB-231 cells exhibited the lowest K+levels, suggesting that a reduced intracellular K+level may favor aggressive behavior and survival. To validate this observation from the sensor-based K+measurements, the study performed inductively coupled plasma mass spectrometry (ICP-MS) on MCF10A, MCF-7, and MDA- MB-231 cells. The results showed the highest K+concentration in MCF10A and the lowest in MDA-MB-231 (FIG.14), confirming the reliability and accuracy of the sensor in quantifying intracellular K+levels across cell types.

[0183] The upregulation of potassium efflux channels has previously been associated with cancer progression and increased tumor aggressiveness. Specifically, Eag1 (Kv10.1), hERG1 (KCNH2), and KCNMA1 (the large-conductance calcium-activated K+channel, BK) are frequently overexpressed in various cancers, including breast cancer

[0076] . These channels facilitate enhanced K+efflux, which contributes to membrane hyperpolarization, increased proliferation, and migration of cancer cells. For instance, Kv10.1 has been shown to be upregulated in multiple tumor types and is linked to poor prognosis

[0077] , while hERG1 expression promotes a pro-oncogenic signaling environment and is associated with metastatic potential [78,79]. KCNMA1 is similarly implicated in promoting invasiveness and epithelial- to-mesenchymal transition

[0080] . While these reports indicate that K+channel dysregulation in malignancy is associated with elevated activity of K+efflux channels, the observation of lower intracellular K+levels in more aggressive tumor cells by the DNAzyme sensor provided a firm experimental confirmation in real time.

[0184] Understanding how cancer cells establish K+-rich microenvironments during tumor progression is crucial. It is widely accepted that K+accumulation at tumor sites primarily results from the release of K+following extensive necrotic tumor cell death due to nutrient deprivation [81,82]. K+imaging revealed that breast cancer cell lines maintain lower intracellular K+levels than non-tumorigenic cells from the same tissue, with the lowest levels observed in metastatic cells. Despite MDA-MB-231 and MCF-7 cells being cultured in a higher K+medium (5.4 mM) compared to MCF-10A cells (4.2 mM), they exhibited lower intracellular K+concentrations. This result suggests that metastatic cells may export K+with higher rate during progression, contributing to high microenvironmental K+in addition to well accepted K+release due to necrosis in cancer cells. To lend support to this suggestion, the study evaluated the efficiency of K+export in various breast tissue cell types by measuring the extracellular K+concentration after incubating the cells in a K+-free Hanks’ Balanced Salt Solution (HBSS) (FIG.13C). Following a 2-hour incubation, the extracellular buffer from the MCF10A cells showed the lowest K+concentration, suggesting minimal efficiency in the K+^Attorney Docket No.10046-637WO1 export. In comparison, MDA-MB-231 cells exhibited reduced K+export relative to MCF7 cells, likely caused by either reduced export activity or a lower intracellular K+concentration.

[0185] By quantifying both changes in intracellular K+homeostasis and the release of K+from these cell lines, these findings suggest that cancerous cells export K+at higher rates during progression, contributing to elevated extracellular K+levels in the tumor microenvironment. This increased K+efflux aligns with previous reports of altered K+channel expression and function in cancer cells, leading to enhanced K+release compared to normal tissue cells. For instance, the outward-flowing K+channels Kv10.1 and Kv11.1 are overexpressed in multiple cancer cell lines, including MCF-7 and MDA-MB-231 breast cancer cells [77,78,83]. Additionally, the Kv1.3 K+efflux channel, which plays a role in cell proliferation and survival, is upregulated in triple-negative breast cancers

[0084] . Therefore, the increase in K+levels in the tumor microenvironment during metastasis may not merely result from passive leakage due to cell death but rather include an active and complementary process driven by differential K+efflux rates in cancer cells during progression.

[0186] Roles of Environmental K+in Anti-Cancer Compounds Resistance: The tumor microenvironment is characterized by abnormally high K+levels, which can be over 40 mM in solid tumors and impair immune functions and aid cancer cell survival [10,85]. It is intriguing to consider whether cancer cells exploit K+-rich environments to disrupt anti-cancer treatment efficacy. The study explored the effects of extracellular K+levels on preclinical anti-cancer compounds by evaluating the efficacy of K+channel-dependent anti-cancer molecule ML133, a Kir2.1 K+channel inhibitor that blocks inward K+flow

[0086] , on MDA-MB-231 cells cultured under high and low K+conditions.

[0187] To explore the impact of extracellular K^ on the cellular response to ML133, the study quantified the viability of MDA-MB-231 cells under varying extracellular K^ conditions. At a fixed ML133 concentration, cells cultured in high-K^ medium (40 mM, mimicking tumor interstitial fluid) exhibited significantly reduced cell death compared to those in standard medium (FIG. 15A). Conversely, lowering extracellular K^ levels by chelating K^ with 18- crown-6 enhanced ML133-induced cytotoxicity, indicating that ML133’s anti-cancer activity is inversely correlated with environmental K^ concentration.

[0188] It was hypothesized that this protective effect of extracellular K^ is mediated through its influence on intracellular K^ homeostasis. To test this hypothesis, the study increased ML133 concentrations and observed a dose-dependent decrease in intracellular K^ (FIGS. 15B-15C). In contrast, cells cultured in high-K^ media maintained elevated intracellular K^ levels (FIGS. 15D-15E), likely due to compensatory K^ influx through ^Attorney Docket No.10046-637WO1 alternative potassium channels not inhibited by ML133. In the physiological context, high extracellular K^ levels in the tumor microenvironment are known to arise from necrotic cell death and leakage of intracellular contents, which may help sustain intracellular K^ levels and promote resistance to K^ channel inhibitors such as ML133.

[0189] Connecting chemoresistance with elevated microenvironmental K+is a compelling avenue of investigation. The study further tested if this K+-dependent anti-cancer treatment resistance is generalizable and applies to other anti-cancer compounds affecting membrane potentials, given that environmental ion concentration influences these potentials. The study evaluated Amiodarone, a clinically approved antiarrhythmic agent, which has been repurposed in preclinical and clinical studies for potential anti-cancer applications, though it is not yet approved for cancer therapy

[0087] . As shown in FIG. 16A, while adding increasing concentrations of Amiodarone reduced the MDA-MB-231 cell viability, supplementing K+to the cell culture media made the cells more viable, suggesting that the elevated K+levels in the culture media reduced the efficacy of Amiodarone in inhibiting MDA-MB-231 cell growth, similar to the effects observed with ML133 treatment. In contrast, decreasing the K+concentration in the cell media by adding the 18-crown-6 K+chelator drastically reduced the cell viability. To find out the K+levels under these treatments, the study applied the 10-9 DNAzyme sensor to the system. As shown in FIGS. 16B-16C, when the drug concentration increased, observed significant decreases in intracellular K+levels were observed. Adding 40 mM K+to the cell culture media was able to compensate for intracellular K+decreases and rescue cell viability, as shown by K+fluorescence imaging and quantification (FIGS. 16D- 16E). Together these results suggest that a high K+level in the tumor microenvironment confer resistance across multiple anti-cancer compounds.

[0190] Given the above finding that a high K+level in the tumor microenvironment confers treatment resistance across multiple anti-cancer compounds, the study explored ways to overcome such treatment resistance through reversing the compensation of intracellular K+levels by disrupting inward K+flow into metastatic cancer cells. Thus, the study tested if inhibiting the intake of K+through the Kir2.1 channel will sensitize MDA-MB-231 cells to the Amiodarone treatment under high environmental K+. As shown in FIGS. 16F-16G, in the presence of a high (40 mM) K+in the cell culture media, adding 30 µM ML133 results in minimal growth inhibition of MDA-MB-231 cells, with a viability of 92.3%. Similarly, treating the cells with 20 µM Amiodarone resulted in 76.8% viability. However, the combination of 30 µM ML133 with 20 µM Amiodarone markedly reduced cell viability to 38.7%. This co- treatment effectively reversed the high-K+microenvironment-induced treatment resistance, ^Attorney Docket No.10046-637WO1 restoring cell sensitivity to a level similar to that observed with 20 µM Amiodarone in the absence of elevated environmental K+(41.5% viability). Quantification of K+imaging in FIG. 16H demonstrated that the intracellular K+concentration is significantly lower when the two compounds are combined compared to when each compound is applied individually. The combination of these two compounds reversed the increase in intracellular K+levels induced by extracellular K+supplementation, bringing them back to the levels observed in cells treated with amiodarone alone, without additional K+.

[0191] To assess the synergistic effect of ML133 and Amiodarone, the study applied the Bliss Independence Model

[0088] . Treatment with 30^µM ML133 and 20^µM Amiodarone individually resulted in 7.7% and 23.2% inhibition of cell viability, respectively. Combined treatment produced 61.3% inhibition, substantially exceeding the 29.1% predicted by the Bliss independence model, indicating a synergistic interaction. The synergistic reductions in cell viability suggest that targeting K+homeostasis, either by modulating extracellular K+levels with chelators or by inhibiting K+uptake with channel blockers, could enhance the effectiveness of certain types of anti-cancer treatment. These findings support the growing body of recent evidence highlighting the alterations of K+channels in chemoresistance89,90 and indicate that modulating extracellular K+levels or inhibiting K+uptake could be a potential strategy to enhance anti-cancer treatment effectiveness. These results contribute to ongoing efforts to understand ion channel regulation in cancer treatment and provide further insight into how K+homeostasis influences therapeutic outcomes. Given that elevated extracellular K+levels have been observed in multiple solid tumor models, further investigation is valuable in determining whether K+-dependent chemoresistance is a generalizable mechanism across different tumor types and anti-cancer compounds. Future studies could explore additional K+channel inhibitors or combination therapies to further disrupt K+-dependent survival pathways in cancer cells. Moreover, incorporating high-K+conditions that better mimic the tumor microenvironment in anti-cancer drug screening may improve the identification of more effective therapeutic strategies. Conclusion

[0192] Highly selective imaging of K+and the quantitative visualization of intracellular K+within physiologically relevant concentration ranges are essential for understanding the role of K+in biological processes and disease progression. However, achieving both high selectivity and a matched detection range remains technically challenging. To address this limitation, the study selected the first highly K+-specific RNA-cleaving DNAzyme with substantial activity at a broad range of K+concentrations matching intracellular K+levels. The study subsequently ^Attorney Docket No.10046-637WO1 engineered this DNAzyme into a catalytic beacon fluorescent sensor for intracellular K+imaging across various cell lines, including human breast tissue cells. This DNAzyme-based sensor addresses key limitations of previously reported K+sensors, enabling broader biochemical and biomedical applications. First, most small molecule sensors exhibit only moderate selectivity for K^ over Na^ (typically < 5-fold) [17,18], leading to interference in Na^- rich organelles and microenvironments in cells. In contrast, this DNAzyme sensor achieves over 1000-fold selectivity for K^ over Na^ and >100-fold selectivity against other ions, ensuring fluorescence signals reflect K^ concentrations with low interference. Second, while some triazacryptand-based small-molecule and nanoparticle sensors offer improved selectivity, they require complex synthesis and multi-component assembly to correct for pH-dependent biases, limiting accessibility and reproducibility [19–21]. This DNAzyme sensor, in contrast, features a simple design that can be readily synthesized by any DNA synthesis company, enhancing scalability and research accessibility. Finally, genetically encoded K+sensors typically have low dissociation constants (usually Kd < 30 mM) [23–26], which do not align well with the physiological intracellular K^ concentration (140–150 mM), limiting broader applicability. This DNAzyme sensor, with a Kd of ~105 mM and a linear detection range of 21–200 mM, matches intracellular K^ concentrations, making it ideal for studying cellular K^ dynamics in health and disease.

[0193] Utilizing the DNAzyme sensor, the study identified a progressive decline in steady- state K+homeostasis across normal, non-invasive, and invasive breast tissue cells. It was observed that cancer cells regulate K+efflux with varying efficiencies, with tumor cells exhibiting a high rate of K+release, leading to an elevated extracellular K+environment. It would be interesting to explore potential connection with expression levels of outward-flowing K+channels in a future study. By monitoring intracellular K+homeostasis following anti- cancer treatment in both high- and normal-K+environments, the study discovered a compensatory effect of elevated extracellular K+in maintaining intracellular K+levels during chemotherapy. This study highlights the role of extracellular K+accumulation in buffering the effects of anti-cancer compounds and stabilizing intracellular K+levels in more aggressive tumor cells, thereby promoting treatment resistance. These insights establish a crucial link between cancer cell K+homeostasis, tumor microenvironmental K+accumulation, and chemoresistance. This DNAzyme sensor provides a highly selective and physiologically relevant method for studying K+regulation in biological systems, offering a valuable tool to advance understanding of K+-mediated drug resistance and serves as a foundation for future research aimed at developing more effective cancer treatments. ^Attorney Docket No.10046-637WO1 EXAMPLE ASPECTS

[0194] Example 1: A DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when potassium (K+) is not present; and an enzyme strand at least partially complementary to the substrate strand and comprising a catalytic loop; wherein the catalytic loop is capable of cleaving the substrate strand at the cleavage site in the presence of K+, wherein said cleavage activates the detectable signal; wherein the catalytic loop comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof.

[0195] Example 2: The DNAzyme sensor of any examples herein, particularly Example 1, wherein the cleavage site comprises at least one RNA base.

[0196] Example 3: The DNAzyme sensor of any examples herein, particularly Example 2, wherein the cleavage site comprises from 1 to 25 RNA bases.

[0197] Example 4: The DNAzyme sensor of any examples herein, particularly Examples 1-3, wherein the cleavage site is interspersed between two segments of nucleic acid.

[0198] Example 5: The DNAzyme sensor of any examples herein, particularly Example 4, wherein the two segments of nucleic acid have a same length.

[0199] Example 6: The DNAzyme sensor of any examples herein, particularly Example 4, wherein the two segments of nucleic acid have different lengths.

[0200] Example 7: The DNAzyme sensor of any examples herein, particularly Examples 4-6, wherein each of the two segments of nucleic acid comprises from 3 nucleotides to 30 nucleotides.

[0201] Example 8: The DNAzyme sensor of any examples herein, particularly Examples 1-7, wherein the substrate strand and / or the enzyme strand comprise DNA.

[0202] Example 9: The DNAzyme sensor of any examples herein, particularly Examples 1-8, wherein the substrate strand further comprises at least one non-natural nucleic acid.

[0203] Example 10: The DNAzyme sensor of any examples herein, particularly Example 9, wherein the at least one non-natural nucleic acid is a locked nucleic acid (LNA) or a 2’- fluoro arabino nucleic acid (FANA).

[0204] Example 11: The DNAzyme sensor of any examples herein, particularly Examples 1-10, wherein the enzyme strand comprises a K+binding region.

[0205] Example 12: The DNAzyme sensor of any examples herein, particularly Examples 1-11, wherein the catalytic loop comprises SEQ ID NO: 1 or SEQ ID NO: 2. ^Attorney Docket No.10046-637WO1

[0206] Example 13: The DNAzyme sensor of any examples herein, particularly Examples 1-12, wherein the catalytic loop comprises a variant of SEQ ID NO: 1 or a variant of SEQ ID NO: 2.

[0207] Example 14: The DNAzyme sensor of any examples herein, particularly Examples 1-13, wherein the substrate strand comprises SEQ ID NO: 3 or SEQ ID NO: 4; and wherein the enzyme strand comprises SEQ ID NO: 6 or SEQ ID NO: 7.

[0208] Example 15: The DNAzyme sensor of any examples herein, particularly Examples 1-14, wherein the substrate strand comprises a variant of SEQ ID NO: 3 or a variant of SEQ ID NO: 4; and wherein the enzyme strand comprises a variant of SEQ ID NO: 6 or a variant of SEQ ID NO: 7.

[0209] Example 16: The DNAzyme sensor of any examples herein, particularly Examples 1-15, wherein the substrate strand and the enzyme strand are linked together.

[0210] Example 17: The DNAzyme sensor of any examples herein, particularly Example 16, wherein the DNAzyme comprises 80% similarity or more to SEQ ID NO: 10.

[0211] Example 18: The DNAzyme sensor of any examples herein, particularly Examples 16-17, wherein the DNAzyme sensor comprises 90% similarity or more to SEQ ID NO: 10.

[0212] Example 19: The DNAzyme sensor of any examples herein, particularly Examples 1-18, wherein the DNAzyme sensor comprises SEQ ID NO: 10.

[0213] Example 20: The DNAzyme sensor of any examples herein, particularly Examples 1-19, wherein the detectable signal is a fluorophore or a fluorescent dye.

[0214] Example 21: The DNAzyme sensor of any examples herein, particularly Example 20, wherein the detectable signal is Alexa Fluor 647 or Alexa Fluor 488.

[0215] Example 22: The DNAzyme sensor of any examples herein, particularly Examples 1-21, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.

[0216] Example 23: The DNAzyme sensor of any examples herein, particularly Example 22, wherein the detectable signal is indocyanine green, methylene blue, or Evans blue.

[0217] Example 24: The DNAzyme sensor of any examples herein, particularly Examples 1-23, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a complementary end of the enzyme strand.

[0218] Example 25: The DNAzyme sensor of any examples herein, particularly Examples 1-24, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a second end of the substrate strand. ^Attorney Docket No.10046-637WO1

[0219] Example 26: The DNAzyme sensor of any examples herein, particularly Examples 24-25, wherein the quencher is Iowa Black RQ or Iowa Black FQ.

[0220] Example 27: The DNAzyme sensor of any examples herein, particularly Examples 1-26, wherein the catalytic loop has a dissociation constant (Kd) for K+of from about 50 µM to about 200 mM.

[0221] Example 28: A method of detecting K+, the method comprising: a) exposing a sample to the DNAzyme sensor of any examples herein, particularly Examples 1-27; and b) identifying the detectable signal, thereby detecting K+in the sample.

[0222] Example 29: The method of any examples herein, particularly Example 28, further comprising, before step a), providing a reference level of the detectable signal by: i) providing to the sample an inactive DNAzyme sensor, the inactive DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; and an inactive enzyme strand at least partially complementary to the substrate strand comprising at least one mutation; wherein the at least one mutation prevents the inactive enzyme strand from cleaving the substrate strand; and ii) identifying the detectable signal, thereby detecting K+in the sample; wherein the reference level of the detectable signal is used to eliminate background noise in data obtained in step b).

[0223] Example 30: The method of any examples herein, particularly Example 29, wherein the substrate strand comprises SEQ ID NO: 3 or SEQ ID NO: 4; and wherein the inactive enzyme strand comprises SEQ ID NO: 11.

[0224] Example 31: The method of any examples herein, particularly Examples 29-30, wherein the substrate strand comprises a variant of SEQ ID NO: 3 or a variant of SEQ ID NO: 4; and wherein the inactive enzyme strand comprises a variant of SEQ ID NO: 11.

[0225] Example 32: The method of any examples herein, particularly Examples 28-31, further comprising, before step a), annealing the substrate strand and the enzyme strand together.

[0226] Example 33: The method of any examples herein, particularly Examples 28-32, further comprising, after step a) and before step b), exposing the sample to an acoustic signal; wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to the acoustic signal.

[0227] Example 34: The method of any examples herein, particularly Example 33, wherein the acoustic signal is high frequency ultrasound (HIFU).

[0228] Example 35: The method of any examples herein, particularly Examples 28-34, wherein the method is used to quantify K+in the sample. ^Attorney Docket No.10046-637WO1

[0229] Example 36: The method of any examples herein, particularly Examples 28-35, wherein the sample is a cell or tissue sample.

[0230] Example 37: The method of any examples herein, particularly Example 36, wherein the method further comprises spatially identifying K+in the cell or tissue sample.

[0231] Example 38: The method of any examples herein, particularly Examples 28-35, wherein the sample is cell lysate or a biological fluid.

[0232] Example 39: The method of any examples herein, particularly Examples 28-38, wherein the sample is derived from a subject having hyperkalemia or hypokalemia, or wherein the subject has been given medication which causes hyperkalemia or hypokalemia.

[0233] Example 40: The method of any examples herein, particularly Examples 28-39, wherein the sample is derived from a subject having a cancer, an immune disorder, a neural disorder, a cardiovascular disorder, a kidney disorder, a metabolic disorder, or an endocrine disorder.

[0234] Example 41: The method of any examples herein, particularly Examples 28-40, wherein the method is carried out in vivo, ex vivo, or in vitro.

[0235] Example 42: A method of determining usefulness of a test agent in modulating K+, the method comprising: a) exposing the test agent to a sample; b) exposing the sample to the DNAzyme sensor of any examples herein, particularly Examples 1-27; c) identifying the detectable signal, thereby detecting K+in the sample; and d) using said detectable signal to determine an effect of the test agent on K+.

[0236] Example 43: The method of any examples herein, particularly Example 42, wherein the effect of the test agent on K+comprises an increase in K+amount or activity, and wherein the detectable signal is greater than a reference signal produced by the sample not exposed to the test agent.

[0237] Example 44: The method of any examples herein, particularly Example 42, wherein the effect of the test agent on K+comprises a decrease in K+amount or activity, and wherein the detectable signal is lesser than a reference signal produced by the sample not exposed to the test agent.

[0238] Example 45: The method of any examples herein, particularly Examples 42-44, wherein the sample is a cell or tissue sample.

[0239] Example 46: The method of any examples herein, particularly Example 45, wherein the method further comprises spatially identifying K+in the cell or tissue sample.

[0240] Example 47: The method of any examples herein, particularly Examples 42-46, wherein the sample is cell lysate or a biological fluid. ^Attorney Docket No.10046-637WO1

[0241] Example 48: The method of any examples herein, particularly Examples 42-47, wherein the test agent is an anti-cancer agent, a beta-adrenergic agonist, a beta-blockade, an alpha-agonist, a blood pressure medication, a diuretic, an anti-inflammatory agent, an antibiotic, or a blood thinner.

[0242] Example 49: The method of any examples herein, particularly Examples 42-48, wherein the test agent is used to treat hyperkalemia or hypokalemia.

[0243] Example 50: The method of any examples herein, particularly Examples 42-49, wherein the test agent induces hyperkalemia or hypokalemia.

[0244] Example 51: The method of any examples herein, particularly Examples 42-50, wherein the test agent is used to treat a cancer, an immune disorder, a neural disorder, a cardiovascular disorder, a kidney disorder, a metabolic disorder, or an endocrine disorder.

[0245] Example 52: The method of any examples herein, particularly Examples 42-51, wherein the sample is derived from a subject having hyperkalemia or hypokalemia, or wherein the subject has been given medication which causes hyperkalemia or hypokalemia.

[0246] Example 53: The method of any examples herein, particularly Examples 42-52, wherein the sample is derived from a subject having a cancer, an immune disorder, a neural disorder, a cardiovascular disorder, a kidney disorder, a metabolic disorder, or an endocrine disorder.

[0247] Example 54: The method of any examples herein, particularly Examples 42-53, wherein the method is carried out in vivo, ex vivo, or in vitro.

[0248] Example 55: A method of treating and / or preventing dysregulation of K+in a subject in need thereof, the method comprising: a) administering to the subject the DNAzyme sensor of any examples herein, particularly Examples 1-27; b) identifying the detectable signal, thereby detecting K+in the subject; and c) using said detectable signal to trigger a therapeutic event.

[0249] Example 56: The method of any examples herein, particularly Example 55, wherein the subject has hyperkalemia or hypokalemia, or wherein the subject has been given medication which causes hyperkalemia or hypokalemia.

[0250] Example 57: The method of any examples herein, particularly Examples 55-56, wherein the subject has a cancer, an immune disorder, a neural disorder, a cardiovascular disorder, a kidney disorder, a metabolic disorder, or an endocrine disorder.

[0251] Example 58: The method of any examples herein, particularly Examples 55-57, wherein the detectable signal is used to determine timing, dosage, or administration route of a therapeutic agent. ^Attorney Docket No.10046-637WO1

[0252] Example 59: The method of any examples herein, particularly Examples 55-58, wherein the detectable signal is identified by imaging the patient.

[0253] Example 60: The method of any examples herein, particularly Examples 55-59, wherein the detectable signal is identified in a sample of a biological fluid collected from the patient.

[0254] Example 61: The method of any examples herein, particularly Examples 55-60, wherein the therapeutic agent comprises a small molecule, a biologic agent, a peptide, a nucleic acid, radiation, chemotherapy, and / or surgery.

[0255] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein. Reference List (1) Jomova, K.; et al. Essential Metals in Health and Disease. Chemico-Biological Interactions 2022, 367, 110173. (2) Abdul Kadir, L.; et al. Emerging Roles of the Membrane Potential: Action Beyond the Action Potential. Front. Physiol.2018, 9. (3) Snyders, D. J. Structure and Function of Cardiac Potassium Channels. Cardiovascular Research 1999, 42 (2), 377–390. (4) Macknight, A. D. C. Principles of Cell Volume Regulation. Renal Physiology and Biochemistry 2008, 11 (3–5), 114–141. (5) Casey, J. R.; et al. Sensors and Regulators of Intracellular pH. Nat Rev Mol Cell Biol 2010, 11 (1), 50–61. (6) Page, M. J.; et al. Role of Na+and K+in Enzyme Function. Physiological Reviews 2006, 86 (4), 1049–1092. (7) Li, G.-R.; et al. Ion Channels in Mesenchymal Stem Cells from Rat Bone Marrow. Stem Cells 2006, 24 (6), 1519–1528. (8) Yu, S. P. Regulation and Critical Role of Potassium Homeostasis in Apoptosis. Progress in Neurobiology 2003, 70 (4), 363–386. (9) Gurusamy, D.; et al. Novel ‘Elements’ of Immune Suppression within the Tumor Microenvironment. Cancer Immunol Res 2017, 5 (6), 426–433. ^Attorney Docket No.10046-637WO1 (10) Eil, R.; et al. Ionic Immune Suppression within the Tumour Microenvironment Limits T Cell Effector Function. Nature 2016, 537 (7621), 539–543. (11) Vodnala, S. K.; et al. T Cell Stemness and Dysfunction in Tumors Are Triggered by a Common Mechanism. Science 2019, 363 (6434), eaau0135. (12) Chirra, M.; et al. How the Potassium Channel Response of T Lymphocytes to the Tumor Microenvironment Shapes Antitumor Immunity. Cancers 2022, 14 (15), 3564. (13) Ong, S. T.; et al. Extracellular K+Dampens T Cell Functions: Implications for Immune Suppression in the Tumor Microenvironment. Bioelectricity 2019, 1 (3), 169–179. (14) Heintzman, D. R.; et al Microenvironmental Influences on T Cell Immunity in Cancer and Inflammation. Cell Mol Immunol 2022, 19 (3), 316–326. (15) Burg, E. D.; et al. K+Channels in Apoptosis. J Membrane Biol 2006, 209 (1), 3–20. (16) Wang, Z. Roles of K+Channels in Regulating Tumour Cell Proliferation and Apoptosis. Pflugers Arch - Eur J Physiol 2004, 448 (3), 274–286. (17) Zhou, X.; et al. A New Highly Selective Fluorescent K+Sensor. J. Am. Chem. Soc. 2011, 133 (46), 18530–18533. (18) Song, G.; et al. A Highly Selective, Colorimetric, and Environment-Sensitive Optical Potassium Ion Sensor. Chem. Commun.2017, 53 (41), 5602–5605. (19) Anees, P.; et al. Detecting Organelle-Specific Activity of Potassium Channels with a DNA Nanodevice. Nat Biotechnol 2024, 42 (7), 1065–1074. (20) Brown, J. Q.; McShane, M. J. Core-Referenced Ratiometric Fluorescent Potassium Ion Sensors Using Self-Assembled Ultrathin Films on Europium Nanoparticles. IEEE Sensors Journal 2005, 5 (6), 1197–1205. (21) Ahn, M.-S.; et al. Fabrication of a Solution-Gated Transistor Based on Valinomycin Modified Iron Oxide Nanoparticles Decorated Zinc Oxide Nanorods for Potassium Detection. Journal of Colloid and Interface Science 2018, 518, 277–283. (22) Baek, K.; et al. The Design and Evolution of Fluorescent Protein-Based Sensors for Monoatomic Ions in Biology. Protein Engineering, Design and Selection 2021, 34, gzab023. ^Attorney Docket No.10046-637WO1 (23) Wu, S.-Y.; et al. A Sensitive and Specific Genetically-Encoded Potassium Ion Biosensor for in vivo Applications across the Tree of Life. PLOS Biology 2022, 20 (9), e3001772. (24) Torres Cabán, C. C.; et al. Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium Indicators through Structure-Guided and Genome Mining Strategies. ACS Sens. 2022, 7 (5), 1336–1346. (25) Bischof, H.; et al. Novel Genetically Encoded Fluorescent Probes Enable Real-Time Detection of Potassium in vitro and in vivo. Nat Commun 2017, 8 (1), 1422. (26) Shen, Y.; et al. Genetically Encoded Fluorescent Indicators for Imaging Intracellular Potassium Ion Concentration. Commun Biol 2019, 2 (1), 1–10. (27) Russo, P.; et al. Regulation of Potassium Homeostasis . In Encyclopedia of Endocrine Diseases (Second Edition); Huhtaniemi, I., Martini, L., Eds.; Academic Press: Oxford, 2018; pp 552–557. (28) Balkay, L.; et al. Flow Cytometric Determination of Intracellular Free Potassium Concentration. Cytometry 1997, 28 (1), 42–49. (29) Gumz, M. L.; et al. An Integrated View of Potassium Homeostasis. New England Journal of Medicine 2015, 373 (1), 60–72. (30) Zhang, T.; et al. Recent Advances in Aptamer-Based Biosensors for Potassium Detection. Analyst 2023, 148 (21), 5340–5354. (31) Shi, C.; et al. An Aptamer-Based Fluorescent Biosensor for Potassium Ion Detection Using a Pyrene-Labeled Molecular Beacon. Analytical Biochemistry 2010, 400 (1), 99–102. (32) Chen, Z.; et al. Colorimetric Detection of Potassium Ions Using Aptamer- Functionalized Gold Nanoparticles. Analytica Chimica Acta 2013, 787, 189–192. (33) Breaker, R. R.; Joyce, G. F. A DNA Enzyme That Cleaves RNA. Chemistry & Biology 1994, 1 (4), 223–229. (34) Li, J.; Lu, Y. A Highly Sensitive and Selective Catalytic DNA Biosensor for Lead Ions. J. Am. Chem. Soc.2000, 122 (42), 10466–10467. ^Attorney Docket No.10046-637WO1 (35) Lake, R. J.; et al. DNAzymes as Activity-Based Sensors for Metal Ions: Recent Applications, Demonstrated Advantages, Current Challenges, and Future Directions. Acc. Chem. Res.2019, 52 (12), 3275–3286. (36) Mao, X.; et al. Activity Modulation and Allosteric Control of a Scaffolded DNAzyme Using a Dynamic DNA Nanostructure. Chemical Science 2016, 7 (2), 1200–1204. (37) Lehman, N.; Joyce, G. F. Evolution in vitro of an RNA Enzyme with Altered Metal Dependence. Nature 1993, 361 (6408), 182–185. (38) Zhou, W.; et al. Metal Sensing by DNA. Chem. Rev.2017, 117 (12), 8272–8325. (39) Breaker, R. R.; Joyce, G. F. Inventing and Improving Ribozyme Function: Rational Design versus Iterative Selection Methods. Trends in Biotechnology 1994, 12 (7), 268–275. (40) Joyce, G. F. Evolution of Catalytic Function. Pure Appl Chem 1993, 65 (6), 1205– 1212. (41) Qian, R.-C.; et al. Metal-Dependent DNAzymes for Cell Surface Engineering and Intracellular Bioimaging. In DNA Nanotechnology for Cell Research; John Wiley & Sons, Ltd, 2024; pp 141–168. (42) Wu, Y.; et al. Photocaged Functional Nucleic Acids for Spatiotemporal Imaging in Biology. Current Opinion in Chemical Biology 2020, 57, 95–104. (43) Khan, S.; et al. DNAzyme-Based Biosensors: Immobilization Strategies, Applications, and Future Prospective. ACS Nano 2021, 15 (9), 13943–13969. (44) Cozma, I.; et al. DNAzymes as Key Components of Biosensing Systems for the Detection of Biological Targets. Biosensors and Bioelectronics 2021, 177, 112972. (45) Adornetto, G.; et al. A General Approach to the Design of Allosteric, Transcription Factor-Regulated DNAzymes. Chemical Science 2015, 6 (7), 3692–3696. (46) Niazov, T.; et al. DNAzyme-Functionalized Au Nanoparticles for the Amplified Detection of DNA or Telomerase Activity. Nano Lett.2004, 4 (9), 1683–1687. (47) Li, J.; et al. In vitro Selection and Characterization of a Highly Efficient Zn(II)- Dependent RNA-Cleaving Deoxyribozyme. Nucleic Acids Research 2000, 28 (2), 481–488. ^Attorney Docket No.10046-637WO1 (48) Xiong, M.; et al. A Semisynthetic Bioluminescence Sensor for Ratiometric Imaging of Metal Ions In vivo Using DNAzymes Conjugated to An Engineered Nano-Luciferase. Angewandte Chemie International Edition 2023, 62 (37), e202308086. (49) Wu, Y.; et al. Ratiometric Detection of Zn2+Using DNAzyme-Based Bioluminescence Resonance Energy Transfer Sensors. Chemistry 2023, 5 (3), 1745–1759. (50) He, Z.-M.; et al. A Targeted DNAzyme-Nanocomposite Probe Equipped with Built-in Zn2+Arsenal for Combined Treatment of Gene Regulation and Drug Delivery. Sci Rep 2016, 6 (1), 22737. (51) Lan, T.; et al. A Highly Selective Lead Sensor Based on a Classic Lead DNAzyme. Chem Commun (Camb) 2010, 46 (22), 3896–3898. (52) Xiao, Y.; et al. Electrochemical Detection of Parts-Per-Billion Lead via an Electrode- Bound DNAzyme Assembly. J. Am. Chem. Soc.2007, 129 (2), 262–263. (53) Liu, J.; et al. A Catalytic Beacon Sensor for Uranium with Parts-per-Trillion Sensitivity and Millionfold Selectivity. Proc Natl Acad Sci U S A 2007, 104 (7), 2056–2061. (54) Lin, Y.; et al. Enzyme-Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells. Angewandte Chemie International Edition 2019, 58 (47), 17061–17067. (55) Xiong, M.; et al. DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells. Angewandte Chemie International Edition 2020, 59 (5), 1891–1896. (56) Heaton, I.; Platt, M. DNAzyme Sensor for the Detection of Ca2+Using Resistive Pulse Sensing. Sensors (Basel) 2020, 20 (20), 5877. (57) Liu, J.; Lu, Y. A DNAzyme Catalytic Beacon Sensor for Paramagnetic Cu2+Ions in Aqueous Solution with High Sensitivity and Selectivity. J. Am. Chem. Soc. 2007, 129 (32), 9838–9839. (58) Huang, P.-J. J.; Liu, J. An Ultrasensitive Light-up Cu2+Biosensor Using a New DNAzyme Cleaving a Phosphorothioate-Modified Substrate. Anal. Chem.2016, 88 (6), 3341– 3347. ^Attorney Docket No.10046-637WO1 (59) Liu, J.; Lu, Y. Rational Design of “Turn-On” Allosteric DNAzyme Catalytic Beacons for Aqueous Mercury Ions with Ultrahigh Sensitivity and Selectivity. Angewandte Chemie International Edition 2007, 46 (40), 7587–7590. (60) Saran, R.; Liu, J. A Silver DNAzyme. Anal. Chem.2016, 88 (7), 4014–4020. (61) Gu, L.; et al. Reselection Yielding a Smaller and More Active Silver-Specific DNAzyme. ACS Omega 2018. (62) Torabi, S.-F.; et al. In vitro Selection of a Sodium-Specific DNAzyme and Its Application in Intracellular Sensing. Proc Natl Acad Sci U S A 2015, 112 (19), 5903–5908. (63) Xiong, Y.; et al. Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets. J. Am. Chem. Soc.2020, 142 (1), 207–213. (64) McGhee, C. E.; et al. DNAzyme-Based Lithium-Selective Imaging Reveals Higher Lithium Accumulation in Bipolar Disorder Patient-Derived Neurons. ACS Cent. Sci. 2021, 7 (11), 1809–1820. (65) Yang, Z.; et al. On-Site Portable Lithium Detection in Mining and Recycling Industries Based on a DNAzyme Fluorescent Sensor. Angewandte Chemie International Edition n / a (n / a), e202413118. (66) Zhou, W.; et al. In vitro Selection of Chromium-Dependent DNAzymes for Sensing Chromium(III) and Chromium(VI). Chemistry – A European Journal 2016, 22 (28), 9835– 9840. (67) Wu, Y.; et al. Simultaneous Fe2+ / Fe3+Imaging Shows Fe3+over Fe2+Enrichment in Alzheimer’s Disease Mouse Brain. Science Advances 2023, 9 (16), eade7622. (68) Huang, P.-J. J.; et al. Distinction of Individual Lanthanide Ions with a DNAzyme Beacon Array. ACS Sens.2016, 1 (6), 732–738. (69) Liu, J. Lanthanide-Dependent RNA-Cleaving DNAzymes as Metal Biosensors. Can. J. Chem.2015, 93 (3), 273–278. (70) Lin, W. T. D.; et al. The Group Trend of Lanthanides Binding to DNA and DNAzymes with a Complex but Symmetric Pattern. Chem. Commun.2014, 50 (80), 11859–11862. ^Attorney Docket No.10046-637WO1 (71) Yang, Z.; et al. Optical Control of Metal Ion Probes in Cells and Zebrafish Using Highly Selective DNAzymes Conjugated to Upconversion Nanoparticles. J. Am. Chem. Soc. 2018, 140 (50), 17656–17665. (72) Qian, R.-C.; et al. Cell Surface Engineering Using DNAzymes: Metal Ion Mediated Control of Cell–Cell Interactions. J. Am. Chem. Soc.2021, 143 (15), 5737–5744. (73) Wang, X.; et al. Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In vivo Using High-Intensity Focused Ultrasound. J. Am. Chem. Soc.2022, 144 (13), 5812–5819. (74) Century, T. J.; et al. The Concentrations of Water, Sodium and Potassium in the Nucleus and Cytoplasm of Amphibian Oocytes. Journal of Cell Science 1970, 7 (1), 5–13. (75) Moore, R. D.; Morrill, G. A. A Possible Mechanism for Concentrating Sodium and Potassium in the Cell Nucleus. Biophysical Journal 1976, 16 (5), 527. (76) Rao, V. R.; et al. Voltage-Gated Ion Channels in Cancer Cell Proliferation. Cancers 2015, 7 (2), 849–875. (77) Luis, E.; et al. The Kv10.1 Channel: A Promising Target in Cancer. Int J Mol Sci 2022, 23 (15), 8458. (78) Breuer, E.-K.; et al. Potassium Channel Activity Controls Breast Cancer Metastasis by Affecting ^-Catenin Signaling. Cell Death Dis 2019, 10 (3), 1–15. (79) Gentile, S. hERG1 Potassium Channel in Cancer Cells: A Tool to Reprogram Immortality. Eur Biophys J 2016, 45 (7), 649–655. (80) Oeggerli, M.; et al. Role of KCNMA1 in Breast Cancer. PLOS ONE 2012, 7 (8), e41664. (81) Mirrakhimov, A. E.; et al. Tumor Lysis Syndrome in Solid Tumors: An up to Date Review of the Literature. Rare Tumors 2014, 6 (2), 68–76. (82) Liu, Z.; Jiao, D. Necroptosis, Tumor Necrosis and Tumorigenesis. Cell Stress 4 (1), 1– 8. (83) Asher, V.; et al. Eag and HERG Potassium Channels as Novel Therapeutic Targets in Cancer. World Journal of Surgical Oncology 2010, 8 (1), 113. ^Attorney Docket No.10046-637WO1 (84) Teisseyre, A.; et al. Voltage-Gated Potassium Channel Kv1.3 as a Target in Therapy of Cancer. Front. Oncol.2019, 9. (85) Vaupel, P.; Multhoff, G. Accomplices of the Hypoxic Tumor Microenvironment Compromising Antitumor Immunity: Adenosine, Lactate, Acidosis, Vascular Endothelial Growth Factor, Potassium Ions, and Phosphatidylserine. Front. Immunol.2017, 8. (86) Wang, H.-R.; et al. Selective Inhibition of the Kir2 Family of Inward Rectifier Potassium Channels by a Small Molecule Probe: The Discovery, SAR and Pharmacological Characterization of ML133. ACS Chem Biol 2011, 6 (8), 845–856. (87) Steinberg, E.; et al. Low Dose Amiodarone Reduces Tumor Growth and Angiogenesis. Sci Rep 2020, 10, 18034. (88) Foucquier, J.; Guedj, M. Analysis of Drug Combinations: Current Methodological Landscape. Pharmacology Research & Perspectives 2015, 3 (3), e00149. (89) Li, M.; et al. Potassium Channels: Novel Targets for Tumor Diagnosis and Chemoresistance. Front. Oncol.2023,(90) Petroni, G. Targeting Potassium Channels and Autophagy to Defeat Chemoresistance. Molecular & Cellular Oncology 2020, 7 (3), 1745038. ^

Claims

Attorney Docket No.10046-637WO1 CLAIMS 1. A DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when potassium (K+) is not present; and an enzyme strand at least partially complementary to the substrate strand and comprising a catalytic loop; wherein the catalytic loop is capable of cleaving the substrate strand at the cleavage site in the presence of K+, wherein said cleavage activates the detectable signal; wherein the catalytic loop comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof.

2. The DNAzyme sensor of claim 1, wherein the cleavage site comprises from 1 to 25 RNA bases.

3. The DNAzyme sensor of claim 1, wherein the cleavage site is interspersed between two segments of nucleic acid.

4. The DNAzyme sensor of claim 3, wherein each of the two segments of nucleic acid comprises from 3 nucleotides to 30 nucleotides.

5. The DNAzyme sensor of claim 1, wherein the substrate strand and / or the enzyme strand comprise DNA.

6. The DNAzyme sensor of claim 1, wherein the substrate strand further comprises at least one non-natural nucleic acid.

7. The DNAzyme sensor of claim 1, wherein the enzyme strand comprises a K+binding region.

8. The DNAzyme sensor of claim 1, wherein the substrate strand comprises SEQ ID NO: 3 or SEQ ID NO: 4; and wherein the enzyme strand comprises SEQ ID NO: 6 or SEQ ID NO:

7. ^Attorney Docket No.10046-637WO1 9. The DNAzyme sensor of claim 1, wherein the substrate strand and the enzyme strand are linked together.

10. The DNAzyme sensor of claim 9, wherein the DNAzyme comprises 80% similarity or more to SEQ ID NO:

10.

11. The DNAzyme sensor of claim 1, wherein the detectable signal is a fluorophore or a fluorescent dye.

12. The DNAzyme sensor of claim 1, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.

13. The DNAzyme sensor of claim 1, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a complementary end of the enzyme strand; or wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a second end of the substrate strand.

14. The DNAzyme sensor of claim 1, wherein the catalytic loop has a dissociation constant (Kd) for K+of from about 50 µM to about 200 mM.

15. A method of detecting K+, the method comprising: a) exposing a sample to the DNAzyme sensor of claim 1; and b) identifying the detectable signal, thereby detecting K+in the sample.

16. The method of any claim 15, further comprising, before step a), providing a reference level of the detectable signal by: i) providing to the sample an inactive DNAzyme sensor, the inactive DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; and ^Attorney Docket No.10046-637WO1 an inactive enzyme strand at least partially complementary to the substrate strand comprising at least one mutation; wherein the at least one mutation prevents the inactive enzyme strand from cleaving the substrate strand; and ii) identifying the detectable signal, thereby detecting K+in the sample; wherein the reference level of the detectable signal is used to eliminate background noise in data obtained in step b).

17. The method of claim 16, wherein the substrate strand comprises SEQ ID NO: 3 or SEQ ID NO: 4 or a variant thereof; and wherein the inactive enzyme strand comprises SEQ ID NO: 11 or a variant thereof.

18. A method of determining usefulness of a test agent in modulating K+, the method comprising: a) exposing the test agent to a sample; b) exposing the sample to the DNAzyme sensor of claim 1; c) identifying the detectable signal, thereby detecting K+in the sample; and d) using said detectable signal to determine an effect of the test agent on K+.

19. The method of claim 18, wherein the effect of the test agent on K+comprises an increase in K+amount or activity, and wherein the detectable signal is greater than a reference signal produced by the sample not exposed to the test agent; and wherein the effect of the test agent on K+comprises a decrease in K+amount or activity, and wherein the detectable signal is lesser than a reference signal produced by the sample not exposed to the test agent.

20. A method of treating and / or preventing dysregulation of K+in a subject in need thereof, the method comprising: a) administering to the subject the DNAzyme sensor of claim 1; b) identifying the detectable signal, thereby detecting K+in the subject; and c) using said detectable signal to trigger a therapeutic event. ^

Images