Systems and methods for enzyme regulation

Abstract

Disclosed herein are systems for regulating enzyme function using an enzyme inhibitor linked to a target recognition sequence, which releases the inhibitor from the enzyme in the presence of a specific target analyte (e.g., a marker of a disease or disorder), thereby activating the enzyme. These enzymes may generate detectable signals and / or perform therapeutic functions. Also disclosed herein are methods of using these systems for the detection of target analytes and the observation, monitoring, and treatment of diseases and disorders.

Description

SYSTEMS AND METHODS FOR ENZYME REGULATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 662.712, filed June 21, 2024, which is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant no. CHE2404334 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 June 23, 2025, as an .XML file entitled “10046- 626WO1_ST26” created on June 23, 2025. and having a file size of 96,717 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0004] Precise control over protein activity is critical for advancing synthetic biology, therapeutics, biochemical sensing, and related fields. In particular, the ability to modulate enzyme function enables dynamic drug delivery systems, biosynthetic circuits, biocatalysts with emergent properties, programmable metabolic pathways, and sensitive biosensors.

[0005] Traditional approaches to enzy me regulation ty pically rely on protein engineering consisting of directed evolution or computational methods. Other common methods include restricting substrate access or inducing conformational changes. Substrate-restriction strategies often use lipid vesicles, synthetic polymers, or DNA origami to block the active site or chemically cage catalytic residues. For example, DNA origami “nanovaults” have been designed to encapsulate enzymes and release them upon the introduction of a specific DNA key. However. DNA origami structures require the assembly of hundreds of distinct DNA strands, making them complex, expensive, and challenging to scale.

[0006] Thus, there exists a need for improved systems and methods for enzyme regulation. These needs and others are at least partially satisfied by the present disclosure.SUMMARY

[0007] Enzymes can be used for a plethora of various applications both in vivo and in vitro. However, precise control over enzyme activity is necessary to enable the use of these enzymes. For example, certain enzy mes may be beneficial in only certain regions of a cell, tissue, or body. In the case of therapeutic uses, enzy mes are often delivered systemically so that an appropriate dose reaches the target site. However, in such cases, a substantial portion of the enzymes are wasted as they do not perform their desired function in the desired region. Additionally, untargeted delivery can also damage regions of the cell, tissue, or body that do not need the enzyme (e.g., activation of a therapeutic enzyme in a healthy region of the body). Similarly, an enzyme that turns on only in the presence of a specific molecular cue can then be used to detect the said cue.

[0008] As such, the present disclosure addresses these challenges by providing a system which regulates enzyme function and activation. Upon administration, the system uses an inhibitor to lock the enzyme in an “off’ state until it encounters a target analyte (e.g., a specific nucleic acid sequence, small molecule, protein, or metal ion or a protease), which then causes the system to disengage the enzyme and allow the enzyme to become active. By selecting a relevant target analyte - for example, a specific marker of a disease or disorder - the system will only release the activated enzy me in response to the presence of the target analyte. In a therapeutic context, this prevents off target activation of the enzy me in healthy tissues and concentrates activated enzymes in the region most affected by the disease or disorder (i.e., where the concentration of the target analyte is highest). Beyond a therapeutic context, this allows for the precise activation of an enzyme in response to a user-defined cue. Furthermore, this system, when used with an enzyme which produces or triggers a detectable signal, can also be highly beneficial for detecting specific target analytes - either regionally or in a sample - as the enzyme would only become activated (and, subsequently, generate a detectable signal) if / where the target analyte is present.

[0009] XXX

[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 1 is a general scheme showing the mechanism of action of SMDTs.

[0012] FIGURES 2A-2D depict thrombin structure and activity. FIG. 2A shows the structure of thrombin bound to its aptamers A15 and A29 (PDB: 5EW1). FIG. 2B Timedependent absorbance plots showing the activity of thrombin in the absence and presence of monovalent and bivalent aptamers. FIG. 2C is a bar plot corresponding to FIG. 2B showing corresponding % activity of thrombin at the 16 min time point. Photographs shown correspond to solutions containing active and inhibited thrombin. FIG. 2D shows % activity of thrombin in the presence of vanous SMDTs.

[0013] FIGURE 3 depicts FIFTA displaying the effect of thrombin concentration on clotting time in a 15-minute time frame in the absence of SMDTs. At / A0is calculated by dividing the absorbance at time t (At) by the absorbance at time 0 (Ao).

[0014] FIGURES 4A-4B depict the effect of increasing A15 (FIG. 4A) and A29 (FIG. 4B) concentrations on T’s clotting activity as measured via FIFTA. High concentrations of A15 lead to inhibition of thrombin, whereas A29 does not effect thrombin activity regardless of concentration.

[0015] FIGURES 5A-5B depict a native PAGE gel showing a comparative assessment of the binding interaction between T and Al 5, A29, and R15. All components are present at 400 nM concentration. FIG. 5A depicts the gel imaged using the GelRed™ fluorescence channel. FIG. 5B depicts the Gel imaged after silver staining. Free T is not visible in the GelRed™ channel but appears faintly after silver staining. A distinct band corresponding to a T-R15 complex is observed (marked with a box), whereas no similar bands are seen for T-A15 or T- A29. These results suggest that R15 associates with T more strongly than Al 5 or A29 under the conditions tested.

[0016] FIGURES 6A-6B depict a native PAGE gel demonstrating the binding of T to various SMDT variants, imaged using the GelRed™ fluorescence channel (FIG. 6A) and silver staining (FIG. 6B). Lanes 1 -10 and lanes 1 1 -19 were run on two separate gels; they are presented together here to enable visual comparison of T mobility shifts upon binding to SMDTs of progressively increasing molecular mass. DNA and protein ladders were included to align and validate band positions across the two gels. In each case, a distinct new band emerges upon mixing T with an SMDT variant, consistent with the formation of a T-SMDT complex. All components were used at 200 nM concentration. Free T is not visible in the GelRed™ channel but appears faintly after silver staining.

[0017] FIGURES 7A-7B show electrophoretic mobility shift of Aptamer 29 (A29) upon binding to thrombin at varying concentrations. Lane 1: DNA ladder. Lane 2: A29 400nM. Lanes 3-8: Thrombin (at increasing concentrations: 200nM, 400nM, 600nM, 900nM, 1200nM,and 1600nM) + A29 400nM, Lane 9: Thrombin 1200nM without A29, Lane 10: Protein ladder. A concentration-dependent mobility shift was observed in lanes 3-8, indicating specific complex formation between A29 and thrombin. No shift occurred for A29 alone (lane 2) or thrombin alone (lane 9). Colocalization of DNA and protein signals confirms a stable aptamerthrombin complex. The band intensities were used to estimate a binding affinity' of ~28 pM. Note that EMSA-derived affinities reflect relative binding within the gel matrix and may differ from solution-phase values due to buffer differences.

[0018] FIGURES 8A-8F depict reactivation experiments. FIG. 8A is a scheme showing reactivation of thrombin with nucleic acid cues. FIG. SB shows reactivation of R10-R40 with complementary nucleic acids. FIG. 8C shows concentration-dependent reactivation of R15. FIG. 8D shows the effect of mismatches on thrombin reactivation. The system shows highest reactivation only in the presence of the full complement, indicating the specificity of the system. FIG. 8E is a scheme showing the effect of complements of different lengths on thrombin reactivation. FIG. 8F shows data displaying reactivation of thrombin when complements of different lengths are added. Complement lengths of 40-nt and above confer the rigidity required to reactivate tngger the SMDT to release active thrombin.

[0019] FIGURE 9 depicts the reactivation of T-SMDT complexes R50 and R60 with complementary' nucleic acid sequences (C50 and C60). The addition of complementary' strands restored thrombin activity, as indicated by the increase in ratio AS / AFconfirming sequencespecific hybridization-mediated reactivation.

[0020] FIGURE 10 depicts the reactivation of T-R15 complex with a 15-nt RNA that is complementary' to the recognition moiety'. The ratio AS / AFrefers to the absorbance of the sample (4S) relative to that of fibrinogen (AF) at the same time-point (16 min). Since fibrinogen absorbance remains constant over time, an AS / AFvalue greater than 1 indicates the presence of active thrombin.

[0021] FIGURES 11A-11K depict additional reactivation and inhibition experiments. FIG. 11A is a scheme showing reactivation of thrombin-SMDT complexes with non-nucleic molecular cues. Structure A is SEQ ID NO: 23, structure B is SEQ ID NO: 22, and structure C is SEQ ID NO: 26. FIG. 11B shows inhibition of thrombin with R-c-Myc. FIG. 11C shows reactivation of R-c-Myc-thrombin complex with c-Myc. FIG. 11D shows inhibition of thrombin with R-PDGF. FIG. HE shows reactivation of R-PDGF-thrombin complex with PDGF. FIG. HF shows inhibition of thrombin with R-TBP. FIG. 11G shows reactivation of R-TBP-thrombin complex with TBP. FIG. 11H shows the effect of concentration of TBP onthrombin reactivation. FIG. Ill shows the specificity' of thrombin reactivation to non-target molecular cues. FIG. 11J shows inhibition of thrombin with R-Kan. FIG. 11K shows reactivation of R-Kan-thrombin complex with Kan.

[0022] FIGURES 12A-12B depict the structure of SMDT-c-Myc with 0-nt spacer (FIG. 12A, SEQ ID NO: 24) or 3-nt spacer (FIG. 12B, SED ID NO: 25) in R domain. These designs did not allow for profound thrombin inhibition due to suboptimal positioning of Al 5 and A29, illustrating the criticality of spacer length in SMDT design.

[0023] FIGURE 13 depicts the effect of increasing nucleotide spacer from 0-nt to 3-nt on inhibitory' ability7of c-Myc SMDT. Increasing the spacer length yielded increased inhibition.

[0024] FIGURES 14A-14F depict the use of the R-Sub. FIG. 14A is a scheme showing mechanism of action of R-Sub (SEQ ID NO: 28). FIG. 14B shows inhibition of thrombin with R-Sub +E. FIG. 14C shows reactivation of thrombin-R-Sub + E complex in the presence of Mn2+. FIG. 14D is a scheme showing mechanism of action of R-RNase (SEQ ID NO: 29). FIG. 14E shows the effect of RNase concentration on thrombin reactivation. FIG. 14F shows the effect of RNase on thrombin-R15 reactivation.

[0025] FIGURE 15 depicts the structure of thrombin bound to A15 and A29 (PDB ID: 5EW1). Distance between 3’ of A15 and 5’ end of A29.

[0026] FIGURE 16 depicts the geometric model used to estimate minimum DNA linker length.

[0027] FIGURE 17 depicts a gel demonstrating the interaction between R50 (at a fixed concentration of 200nM) with thrombin at different concentrations (ranging from 25nM to 800nM). Lane 1: DNA ladder, Lane 2: R50 200nM, Lanes 3-8: Thrombin (at concentrations of 25nM, 50nM, lOOnM, 200nM. 400nM, and 800nM) + R50 200nM, Lane 9: Thrombin 800nM without R50. Lane 10: Protein ladder. A progressive mobility shift in lanes 3-8 with increasing thrombin concentrations indicates concentration-dependent formation of R50-thrombin complexes. No shift was seen for R50 alone (lane 2), and thrombin alone (lane 9) showed no DNA signal, confirming specificity7. Silver staining of the same gel (Figure S8B) verified the presence of thrombin in the shifted bands, further supporting complex formation. Band intensities were used to calculate a binding affinity of ~11 nM for R50 under native conditions.

[0028] FIGURE 18 depicts a native PAGE gel demonstrating the cleavage activity of DNAzyme-SMDT (R-Sub + E) in the presence of Mn2+. All DNA components are prepared at 100 nM. Mn2+is at 100 pM. The SMDT can be seen in the (R-Sub + E) lane as a higher mass band. When treated with 100 pM Mn2. this band decreases in intensity as R-Sub is cleaved by Mn2+.

[0029] FIGURE 19 depicts a gel confirming reactivation of R-RNase / thrombin inactive complex with RNase A.

[0030] FIGURE 20 depicts an example probe structure for detecting active proteases. The target recognition sequence is CGGGFGSQLVASGGGK (SEQ ID NO: 55).

[0031] FIGURE 21 is a schematic of protease activity sensing through thrombin generated turbidity. The target recognition sequence is CGGGFGSQLVASGGGK (SEQ ID NO: 55).

[0032] FIGURE 22 depicts step-wise synthesis of the DNA-peptide-DNA triblock.

[0033] FIGURE 23 depicts UV-Vis and gel electrophoresis characterization of the triblock.

[0034] FIGURES 24A-24B depict inhibition and reactivation using the triblock. The target recognition sequence is CGGGFGSQLVASGGGK (SEQ ID NO: 55).

[0035] FIGURE 25 depicts the limit of detection and selectivity of the triblock.DETAILED DESCRIPTION

[0036] 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. 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

[0037] 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:

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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 than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. "about x, y, z. or less7and should be interpreted to include the specific ranges of "about x’, "about y7, and ‘about z’ as well as the ranges of ‘less than x’, less than y7, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x7, ‘abouty’, 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 ‘x7and ‘y’ are numerical values, includes “about ‘x’ to about "y7”.

[0042] 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 subranges (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.

[0043] 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.

[0044] 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 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.

[0045] 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 orcondition. 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.

[0046] 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.

[0047] 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 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.

[0048] As used herein, the term “prophylactically effective amount" refers to an amount effective for preventing onset or initiation of a disease or condition.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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. Those in need of treatment (subj ects 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.

[0053] 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.

[0054] 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.

[0055] 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).

[0056] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole. 110).

[0057] A “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability.

[0058] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity7to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and / or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereol).

[0059] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V). tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, 0-alanine, 0-Amino-propionic acid, allo-Hydroxylysine acid. 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid. 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid. 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0060] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g.. attachment of myristate, a C14 saturated acid), palmitoylation (e g., attachment of palmitate, a C16 saturated acid), alk lation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g.. the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a non enz matic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g.. of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0061] 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).

[0062] 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 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.

[0063] 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.

[0064] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N- terminus. the C-terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0065] 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 arein-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).

[0066] 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.

[0067] 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.

[0068] Nucleic acid sequences that do not show7a 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.

[0069] “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.

[0070] 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. 1 3, 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.

[0071] “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.

[0072] “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.SYSTEMS FOR REGULATING ENZYME FUNCTION

[0073] Disclosed herein is a system for regulating enzyme function, the system including: a) an enzyme; b) an inhibitor of said enzyme; and c) a target recognition sequence linked to the inhibitor, wherein, when the target recognition sequence interacts with a target analyte, the inhibitor releases the enzyme and the enzyme is activated. The target recognition sequence and the inhibitor form a target recognition sequence-inhibitor construct (for example, the “Single Molecule DNA Tweezer (SMDT)” shown in FIG. 1A) which surrounds and blocks activation of the enzy me until the target analyte (for example, the “molecular cues” shown in FIG. IB) interacts with the target recognition sequence and causes the inhibitor to release the enzyme.

[0074] As described herein, a “target analyte” refers to any moiety which triggers activation of the enzyme. In some aspects, the target analyte may be a nucleic acid, peptide, oligonucleotide, antibody, small molecule, protease, enzyme, microRNA, carbohydrate, or any other molecule which can interact with the target recognition sequence. In some aspects, the target analyte can indicate a disease or disorder, for example, cancer, bacterial infection, viral infection, fungal infection, parasitic infection, neurodegenerative disorders, cardiovasculardiseases, liver and metabolic disorders, inflammatory conditions, renal and pulmonary diseases, or lysosomal storage disorders.

[0075] Similarly, a “target recognition sequence” refers to any one or more components in the system which interacts with the target analyte and induces or enhances activation of the enzy me. For example, in some aspects, interaction between the target recognition sequence and the target analyte can induce a conformational change in the inhibitor, thereby causing the inhibitor to release the enzyme. For example, in some aspects, the effective length or flexibility of the target recognition region can be altered by binding or cleavage, meaning the acceptable distance between the inhibitor, the enzy me, and, when present, the enzyme binding moiety is altered, disrupting the ability of the inhibitor and / or enzyme binding moiety to bind and / or inhibit the enzyme. In some aspects, the target recognition sequence can include nucleic acid (e.g., DNA, RNA, modified nucleic acids, or a combination thereof), an aptamer, a DNAzyme, a carbohydrate, or a peptide sequence.

[0076] Interactions between the target analyte and the target recognition sequence can be achieved via several different mechanisms depending on the specific target analyte. In some aspects, the target analyte can bind to the target recognition sequence. For example, in some aspects, the target recognition sequence can include a first nucleic acid sequence, and the target analyte can include a second nucleic acid sequence that is at least partially complementary to the first nucleic acid sequence. In other aspects, the target recognition sequence can include an aptamer, a DNAzyme, or peptide, and the target analyte can be a small molecule, ion, or peptide.

[0077] In some aspects, the target analyte can cleave the target recognition sequence. For example, in some aspects, the target recognition sequence can include a peptide sequence, and the target analyte can be a protease. In other aspects, the target recognition sequence can include a nucleic acid sequence, and the target analyte can be RNase or DNase.

[0078] In some aspects, the target analyte can induce a conformational change in the target recognition sequence.

[0079] It is understood that the target analyte may interact with the target recognition sequence via any one or more of the ways described herein. For example, in some aspects, the target analyte can bind to and cleave the target recognition sequence. In other aspects, the target analyte can bind to and induce a conformational change in the target recognition sequence. In yet other aspects, the target analyte can induce a conformational change in and cleave the target recognition sequence. In yet still other aspects, the target analyte can bind to, induce a conformational change in, and cleave the target recognition sequence.

[0080] In some aspects, the target recognition sequence can include a nucleic acid sequence. In some aspects, the target recognition sequence can include a single stranded nucleic acid. In some aspect, the target recognition sequence can include DNA, RNA, modified nucleic acids, or any combination thereof. In some aspects, the nucleic acid sequence can be an aptamer or a DNAzy me.

[0081] In some aspects, the target recognition sequence can include about 10 nucleotides or more (e.g., about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more). In some aspects, the target recognition sequence can include about 60 nucleotides or less (e.g.. about 55 or less, about 50 or less, about 45 or less, about 40 or less, about 35 or less, about 30 or less, about 25 or less, about 20 or less, about 15 or less, about 10 or less). The target recognition sequence 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 target recognition sequence can include from about 10 to about 60 nucleotides (e.g., from about 15 to about 55, from about 20 to about 50, from about 25 to about 45, from about 30 to about 40, from about 10 to about 35, from about 15 to about 30. from about 20 to about 25, from about 35 to about 60, from about 40 to about 55, from about 45 to about 50).

[0082] In some aspects, the target recognition sequence can include about 80% similarity' or more (e.g., about 81% similarity or more, about 82% similarity or more, about 83% similarity or more. about 84% similarity or more, about 85% similarity or more, about 86% similarity or more, about 87% similarity or more, about 88% similarity or more, about 89% similarity or more, about 90% similarity or more, about 91% similarity or more, about 92% similarityor more. about 93% similarity' or more, about 94% similarity or more, about 95% similarity or more. about 96% similarity or more, about 97% similarity or more, about 98% similarity or more, about 99% similarity' or more, about 100% similarity or more) to any one of SEQ ID NOs: 3-9, 22-26, or 29.

[0083] In some aspects, the target recognition sequence can be a DNAzyme including an enzyme strand and a substrate strand. In some aspects, the enzyme strand can include about 80% similarity or more (e.g.. about 81% similarity' or more, about 82% similarity or more. about 83% similarity' or more, about 84% similarity or more, about 85% similarity or more, about 86% similarity' or more, about 87% similarity' or more, about 88% similarity7or more, about 89% similarity' or more, about 90% similarity or more, about 91% similarity or more, about 92% similarity or more, about 93% similarity or more, about 94% similarity or more, about 95% similarity or more, about 96% similarity or more, about 97% similarity or more,about 98% similarity or more, about 99% similarity or more, about 100% similarity or more) to SEQ ID NO: 27 and the substrate strand can include about 80% similarity or more (e.g., about 81% similarity or more, about 82% similarity or more, about 83% similarity or more, about 84% similarity or more, about 85% similarity or more, about 86% similarity or more, about 87% similarity or more, about 88% similarity or more, about 89% similarity or more, about 90% similarity or more, about 91% similarity or more, about 92% similarity or more, about 93% similarity or more, about 94% similarity or more, about 95% similarity or more. about 96% similarity or more, about 97% similarity or more, about 98% similarity or more, about 99% similarity or more, about 100% similarity or more) to SEQ ID NO: 28. In some aspects, the target recognition sequence can include the enzy me strand, the substrate strand, or both.

[0084] In some aspects, the target recognition sequence can include a peptide sequence. In some aspects, the target recognition sequence can include a combination of one or more nucleic acid sequences and one or more peptide sequences.

[0085] In some aspects, the target recognition sequence can include about 3 or more amino acids (e.g.. about 4 or more, about 5 or more, about 6 or more, about 7 or more, about 8 or more, about 9 or more, about 10 or more, about 11 or more, about 12 or more, about 13 or more, about 14 or more, about 15 or more, about 16 or more, about 17 or more, about 18 or more, about 19 or more, about 20 or more, about 21 or more, about 22 or more, about 23 or more, about 24 or more, about 25 or more, about 26 or more, about 27 or more, about 28 or more, about 29 or more, about 30 or more). In some aspects, the target recognition sequence can include about 30 or less amino acids (e.g., about 29 or less, about 28 or less, about 27 or less, about 26 or less, about 25 or less, about 24 or less, about 23 or less, about 22 or less, about 21 or less, about 20 or less, about 19 or less, about 18 or less, about 17 or less, about 16 or less, about 15 or less, about 14 or less, about 13 or less, about 12 or less, about 11 or less, about 10 or less, about 9 or less, about 8 or less, about 7 or less, about 6 or less, about 5 or less, about 4 or less, about 3 or less). The target recognition sequence can include any number of amino acids ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the target recognition sequence can include from about 3 to about 30 amino acids (e.g., from about 4 to about 29, from about 5 to about 28, from about 6 to about 27, from about 7 to about 26, from about 8 to about 25, from about 9 to about 24, from about 10 to about 23, from about 11 to about 22, from about 12 to about 21, from about 13 to about 20, from about 14 to about 19, from about 15 to about 18, from about 16 to about 17, from about 3 to about 17, from about 4 to about 16, from about 5 to about 15,from about 6 to about 14, from about 7 to about 13, from about 8 to about 12, from about 9 to about 11, from about 16 to about 30, from about 17 to about 29, from about 18 to about 28, from about 19 to about 27, from about 20 to about 26, from about 21 to about 25, from about 22 to about 24).

[0086] In some aspects, the activated enzyme can produce a detectable signal. For example, in some such aspects, the enzyme can be thrombin. Examples of using a system which uses thrombin to generate a detectable signal are described in further detail below. In other such aspects, the enzyme can be carbonic anhydrase, luciferase, beta-galactosidase, beta-lactamase, lactoperoxidase, horseradish peroxidase, or another suitable enzyme for producing a detectable signal. In other aspects, the enzyme can perform a therapeutic function. For example, in some such aspects, the enzyme can be granzyme B, trypsin, L-asparaginase, pegademase bovine, alglucerase, streptokinase, or another suitable enzyme for performing a therapeutic function. In yet other aspects, the enzyme can produce a detectable signal and perform a therapeutic function.

[0087] In some aspects, the inhibitor can include one or more aptamers, one or more small molecules, one or more peptides, one or more antibodies, or any combination thereof. For example, in some such aspects, the enzyme can be thrombin, and the inhibitor can include about 80% similarity or more (e.g., about 81% similarity or more, about 82% similarity' or more, about 83% similarity or more, about 84% similarity or more, about 85% similarity or more, about 86% similarity or more, about 87% similarity or more, about 88% similarity or more, about 89% similarity or more, about 90% similarity or more, about 91 % similarity or more, about 92% similarity or more, about 93% similarity or more, about 94% similarity or more, about 95% similarity or more, about 96% similarity or more, about 97% similarity or more, about 98% similarity or more, about 99% similarity or more, about 100% similarity) to SEQ ID NO: 1 (thrombin binding aptamer 15).

[0088] In some aspects, the system can further include an enzyme binding moiety linked to the target recognition sequence, and the enzyme binding moiety can include one or more aptamers, one or more small molecules, one or more peptides, one or more antibodies, or any combination thereof. As described herein, an "enzyme binding moiety” refers to any moiety which binds to the enzyme. The enzyme binding moiety may bind to any portion of the enzyme, including to the active site (i.e., the enzyme binding moiety may, in some aspects, contribute to inhibition of the enzy me). In some aspects, the enzyme binding moiety may bind more strongly to the enzyme than the inhibitor and serve to keep the inhibitor anchored to the enzyme. In some aspects, the interaction between the target analyte and the target recognitionsequence may cause the enzyme binding moiety to release the activated enzyme (e.g., by inducing a conformational change as described above for the inhibitor). In other aspects, the enzyme binding moiety may remain bound to the enzyme upon activation of the enzyme. In some such aspects, the enzyme binding moiety may have no effect on the activity' of the enzy me. In other such aspects, the enzy me binging moiety may enhance the activity of the enzyme.

[0089] For example, in some aspects, the enzyme can be thrombin, and the enzyme binding moiety can include about 80% similarity or more (e.g., about 81% similarity or more, about82% similarity7or more, about 83% similarity or more, about 84% similarity or more, about 85% similarity or more, about 86% similarity or more, about 87% similarity or more, about 88% similarity or more, about 89% similarity or more, about 90% similarity or more, about 91% similarity or more, about 92% similarity or more, about 93% similarity or more, about 94% similarity or more, about 95% similarity or more, about 96% similarity or more, about 97% similarity or more, about 98% similarity or more, about 99% similarity' or more, about100% similarity) to SEQ ID NO: 2 (thrombin binding aptamer 29).

[0090] In some aspects, the inhibitor and the enzyme binding moiety can be linked to opposite ends of the target recognition sequence. In some aspects, the inhibitor and / or the enzyme binding moiety' can be linked to the target recognition sequence via a linker. For example, in some such aspects, the linker can include nucleic acid, peptides, and / or others. In other aspects, the inhibitor and / or the enzyme binding moiety can be directly linked to the target recognition sequence.METHODS

[0091] Disclosed herein is a method of regulating enzyme function, the method including providing any of the disclosed systems to a sample or subject. In some aspects, the sample or subject may contain or be suspected to contain the target analyte. In other aspects, the presence of the target analyte in the sample or subject may be unknown. In some aspects, the method may be used to confirm the presence of the target analyte and / or to provide targeted activation of the enzyme within the sample or subject. In other aspects, if the sample or subject does not contain the target analyte, the method can be used to determine the absence of the target analyte and / or to prevent activation of the enzyme in the sample or subject.

[0092] In some aspects, the sample can be a buffer sample, a cell sample, a tissue sample, a biological fluid sample, or a cell lysate sample. In some aspects, the sample can be taken from a subject having or suspected to have a disease or disorder, and the target analyte can indicate a disease or disorder.

[0093] Also disclosed herein is a method of detecting a target analyte in a sample, the method including: a) providing any of the disclosed systems to the sample, wherein the activated enzyme produces a detectable signal; and b) detecting the detectable signal, thereby detecting the target analyte. In some aspects, the sample may contain or be suspected to contain the target analyte. In other aspects, the presence of the target analyte in the sample may be unknown. In some aspects, the method may be used to confirm the presence of the target analyte (i.e.. as indicated by the presence of the detectable signal). In other aspects, if the sample does not contain the target analyte, the method can be used to determine the absence of the target analyte (i.e., as indicated by the absence of the detectable signal).

[0094] In some aspects, the sample can be a buffer sample, a cell sample, a tissue sample, a biological fluid sample, or a cell lysate sample. In some aspects, the sample can be taken from a subject having or suspected to have a disease or disorder.

[0095] In some aspects, the method can further include observing and / or imaging the sample. For example, in some aspects, the method can include visual observation, microscopy, or other imaging techniques or analyses. In some aspects, the method can include determining whether or not the detectable signal is present (i.e., a “yes7’ or “no” binary). Additionally or alternatively, in other aspects, the method can further include quantifying the amount, concentration, or intensity7of detectable signal. For example, in some aspects, quantifying the amount, concentration, or intensity of the detectable signal can be used to determine the amount or concentration of target analyte in the sample).

[0096] In some aspects, the system can be incubated with the sample before the presence or absence of the detectable signal is determined. For example, in some aspects, the system can be incubated with the sample for about 2 minutes or more (e.g., about 3 minutes or more, about 4 minutes or more, about 5 minutes or more, about 10 minutes or more, about 15 minutes or more, about 30 minutes or more, about 45 minutes or more, about 1 hour or more, about 1.5 hours or more, about 2 hours or more, about 3 hours or more, about 4 hours or more, about 6 hours or more, about 8 hours or more, about 10 hours or more, about 12 hours or more, about 16 hours or more, about 20 hours or more, about 24 hours or more). In some aspects, the system can be incubated with the sample for about 24 hours or less (e.g., about 20 hours or less, about 16 hours or less, about 12 hours or less, about 10 hours or less, about 8 hours or less, about 6 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 1.5 hours or less, about 1 hour or less, about 45 minutes or less, about 30 minutes or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less). The system can be incubated with the sample forany duration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the system can be incubated with the sample for from about 2 minutes to about 24 hours (e.g., from about 3 minutes to about 20 hours, from about 4 minutes to about 16 hours, from about 5 minutes to about 12 hours, from about 10 minutes to about 10 hours, from about 15 minutes to about 8 hours, from about 30 minutes to about 6 hours, from about 45 minutes to about 4 hours, from about 1 hour to about 3 hours, from about 1.5 hours to about 2 hours, from about 2 minutes to about 2 hours, from about 3 minutes to about 1.5 hours, from about 4 minutes to about 1 hour, from about 5 minutes to about 45 minutes, from about 10 minutes to about 30 minutes, from about 1.5 hours to about 24 hours, from about 2 hours to about 20 hours, from about 3 hours to about 16 hours, from about 4 hours to about 12 hours, from about 6 hours to about 10 hours).

[0097] In some aspects, the system can be incubated with the sample at a temperature of about 15°C or more (e.g., about 20°C or more, about 25°C or more, about 30°C or more, about 35°C or more, about 40°C or more, about 45°C or more, about 50°C or more, about 55°C or more, about 60°C or more, about 65°C or more, about 70°C or more, about 75°C or more). In some aspects, the system can be incubated with the sample at a temperature of about 75°C or less (e g., about 70°C or less, about 65°C or less, about 60°C or less, about 55°C or less, about 50°C or less, about 45°C or less, about 40°C or less, about 35°C or less, about 30°C or less, about 25°C or less, about 20°C or less, about 15°C or less). The system can be incubated with the sample at a temperature ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the system can be incubated with the sample at a temperature of from about 15°C to about 75°C (e.g., from about 20°C to about 70°C, from about 25°C to about 65°C, from about 30°C to about 60°C, from about 35°C to about 55°C, from about 40°C to about 50°C, from about 15°C to about 45°C, from about 20°C to about 40°C, from about 25°C to about 35°C, from about 45°C to about 75°C, from about 50°C to about 70°C, from about 55°C to about 65°C).

[0098] In one specific example of the disclosure, the method can use thrombin to detect a target analyte in a sample. Thrombin is a serine protease which cleaves soluble fibrinogen into fibrin monomers. These monomers spontaneously polymerize into insoluble fibrin strands, forming a fibrous scaffold (i.e., a blood clot). These clots can be observed with the naked eye and, as such, can serve as a detectable signal that does not require any specialized equipment to detect. Accordingly, in some aspects, the enzyme used in the system can be thrombin, and the method can further include adding fibrinogen to the sample to allow formation of clots (i.e., the detectable signal) if the target analyte is present.

[0099] In some aspects, the system and fibrinogen can be provided to the sample simultaneously. In other aspects, the system can be added to the sample before fibrinogen is added. For example, in some such aspects, the system can be incubated with the sample before addition of fibrinogen in order to allow time for the enzyme to become activated.

[0100] In some aspects, fibrinogen can be added to the sample at a concentration of about 0.5 mg / mL or more (e.g., about 1 mg / mL or more, about 1.5 mg / mL or more, about 2 mg / mL or more, about 2.5 mg / mL or more, about 3 mg / mL or more, about 3.5 mg / mL or more, about 4 mg / mL or more, about 4.5 mg / mL or more, about 5 mg / mL or more). In some aspects, fibrinogen can be added to the sample at a concentration of about 5 mg / mL or less (e.g., about4.5 mg / mL or less, about 4 mg / mL or less, about 3.5 mg / mL or less, about 3 mg / mL or less, about 2.5 mg / mL or less, about 2 mg / mL or less, about 1.5 mg / mL or less, about 1 mg / mL or less, about 0.5 mg / mL or less). Fibrinogen can be added to the sample at any concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, fibrinogen can be added to the sample at a concentration of from about 0.5 mg / mL to about 5 mg / mL (e.g., from about 1 mg / mL to about 4.5 mg / mL, from about1.5 mg / mL to about 4 mg / mL, from about 2 mg / mL to about 3.5 mg / mL. from about 2.5 mg / mL to about 3 mg / mL, from about 0.5 mg / mL to about 3 mg / mL, from about 1 mg / mL to about 2.5 mg / mL, from about 1.5 mg / mL to about 2 mg / mL, from about 2.5 mg / mL to about 5 mg / mL, from about 3 mg / mL to about 4.5 mg / mL, from about 3.5 mg / mL to about 4 mg / mL).

[0101] Also disclosed herein is a method of observing and / or monitoring a disease or disorder in a subject in need thereof, the method including administering to the subject any of the disclosed systems, wherein the target analyte indicates a disease or disorder and the activated enzyme produces a detectable signal. In some aspects, the subject may have or be suspected to have the disease or disorder indicated by the target analyte. In other aspects, the diagnosis of the subject with the disease or disorder indicated by the target analyte may be unknown. In some aspects, the method may be used to confirm the presence of the disease or disorder (e g., as indicated by the presence of the detectable signal). In other aspects, if the subject does not have the disease or disorder, the method can be used to determine the absence of the disease or disorder (e.g., as indicated by the absence of the detectable signal).

[0102] In some aspects, the method can further include observing and / or imaging the subject and / or a sample collected from the subject (e.g., a buffer sample, a cell sample, a tissue sample, a biological fluid sample, or a cell lysate sample). For example, the method can include conducting any suitable medical imaging techniques on the patient (e.g., MRI. PET. X-ray, etc.) or conducting any suitable laboratory’ imaging techniques on the sample (e.g., visualobservation, microscopy, or other imaging techniques or analyses). For example, in some aspects, particularly when a sample is collected from the subject, the method can use a system including thrombin as the enzyme and detect the target analyte via the addition of thrombin and subsequent formation of clots as described above.

[0103] In some aspects, the method can include determining whether or not the detectable signal is present (i.e., a "yes" or “no” binary’)- thereby determining whether or not the subject has the disease or disorder. Additionally or alternatively, in other aspects, the method can further include quantifying the amount, concentration, or intensity7of detectable signal to determine the extent, severity’, or prognosis of the disease or disorder.

[0104] In some aspects, the enzyme may only be activated in a region affected by the disease or disorder. In some such aspects, the method may further include regionally7locating the detectable signal within the subject to determine the regions of the subject affected by the disease or disorder. As one example, in some aspects, the disease or disorder can be cancer or a solid tumor, and the enzy me may be activated only in or near cancerous cells or tumor cells. As another example, in some aspects, the disease or disorder can be a bacterial, viral, fungal, or parasitic infection, and the enzyme may be activated only in or near infected cells.

[0105] In some aspects, upon detection of the detectable signal, one or more treatments for the disease or disorder can be administered to the subject. In some aspects, particularly if the method further includes regionally locating the detectable signal within the subject, the one or more treatments can be administered at and / or surrounding the site of the detectable signal (i.e., the regions of the subject affected by the disease or disorder), thereby allowing for targeted treatment of the disease or disorder and preventing off target administration of the one or more treatments. In some aspects, the activated enzy me may also perform a therapeutic function.

[0106] Also disclosed herein is a method of treating and / or preventing a disease or disorder in a subject in need thereof, the method including administering to the subject any of the disclosed systems, wherein the target analyte indicates a disease or disorder and the activated enzy me performs a therapeutic function. In some aspects, the subject may have or be suspected to have the disease or disorder indicated by the target analyte. In other aspects, the diagnosis of the subject with the disease or disorder indicated by the target analyte may be unknown. In some aspects, the method may be used to provide targeted activation of the enzyme within the subject. In other aspects, if the subject does not have the disease or disorder, the method can be used to prevent activation of the enzyme in the subject.

[0107] In some aspects, the enzyme may only be activated in a region affected by the disease or disorder. As one example, in some aspects, the disease or disorder can be cancer ora solid tumor, and the enzy me may be activated only in or near cancerous cells or tumor cells. As another example, in some aspects, the disease or disorder can be a bacterial, viral, fungal, or parasitic infection, and the enzyme may be activated only in or near infected cells. Accordingly, in some aspects, the activated enzyme may provide its therapeutic effect only at and / or surrounding the regions of the subject affected by the disease or disorder, thereby allowing for targeted treatment of the disease or disorder and preventing off target effects of the enzyme.

[0108] In some aspects, the enzyme can be granzyme B or try psin. In some aspects, the activated enzyme can further produce a detectable signal, which may be detected in the subject or a sample derived from the subject as described above. In some such aspects, the detectable signal may be used to observe and / or monitor the extent, severity, or prognosis of the disease or disorder, and / or to localize the administration of one or more additional treatments as described above.

[0109] In some aspects, in any of the disclosed methods, the enzyme and the target recognition sequence-inhibitor construct can be provided in a ratio of about 1 : 0.5 or more (e.g., about 1: 1 or more, about 1 :2 or more, about 1 :3 or more, about 1 :4 or more, about 1:5 or more, about 1 : 10 or more, about 1 : 15 or more, about 1 :20 or more, about 1 :30 or more, about 1 :40 or more, about 1:50 or more, about 1:60 or more, about 1 :70 or more, about 1 :80 or more, about 1:90 or more, about 1: 100 or more). In some aspects, in any of the disclosed methods, the enzyme and the target recognition sequence-inhibitor construct can be provided in a ratio of about 1 : 100 or less (e g., about 1 : 90 or less, about 1 : 80 or less, about 1 : 70 or less, about 1 :60 or less, about 1 :50 or less, about 1 :40 or less, about 1:30 or less, about 1 :20 or less, about 1 : 15 or less, about 1 : 10 or less, about 1 :5 or less, about 1 :4 or less, about 1:3 or less, about 1:2 or less, about 1 : 1 or less, about 1 :0.5 or less). The enzyme and the target recognition sequenceinhibitor construct can be provided in a ratio ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, in any of the disclosed methods, the enzyme and the target recognition sequence-inhibitor construct can be provided in a ratio of from about 1 :0.5 to about 1: 100 (e g., from about 1 : 1 to about 1 : 90. from about 1 : 2 to about 1 : 80, from about 1 : 3 to about 1:70, from about 1 : 4 to about 1 :60, from about 1 :5 to about 1 :50, from about 1 : 10 to about 1 :40, from about 1 : 15 to about 1:30, from about 1 :0.5 to about 1 :20, from about 1 : 1 to about 1: 15, from about 1 :2 to about 1: 10, from about 1:3 to about 1 :5. from about 1 :20 to about 1 : 100, from about 1 :30 to about 1:90, from about 1 :40 to about 1:80. from about 1:50 to about 1:70).

[0110] In some aspects, any of the disclosed systems may be delivered as naked nucleic acid (unpackaged) or via delivery vehicles. As used herein, the terms "‘delivery vehicle,” “transfer vehicle,” “nanoparticle” or grammatical equivalent, are used interchangeably.[OHl] In some aspects, any of the disclosed systems may be delivered via a single delivery vehicle. In some aspects, any of the disclosed systems may be delivered via one or more delivery vehicles each of a different composition. According to various aspects, suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide- containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphorsilicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery' systems, micelles, emulsions, niosomes, multi-domainblock polymers (vinyl polymers, poly propyl acrylic acid polymers, dynamic poly conjugates), cell- or platelet-derived exosomes. ethosomes, or transfersomes.

[0112] In some aspects, a suitable delivery vehicle can be a lipid nanoparticle. As used herein, “lipid nanoparticles” refer to particles having at least one dimension on the order of nanometers (e.g., 1-1000 nm) and including one or more lipids. In the context of the present invention, a lipid nanoparticle typically serves to transport a desired nucleic acid sequence to a target cell or tissue. The process of incorporation of a desired nucleic acid sequence into a lipid nanoparticle is often referred to as “loading”. The lipids and the nucleic acid sequence can create a self-assembled structure via counterion interactions. The purpose of incorporating a nucleic acid sequence into a transfer vehicle, such as a lipid nanoparticle, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some aspects, a suitable delivery vehicle may be capable of enhancing the stability' of any of the disclosed systems contained therein and / or facilitate the delivery of any of the disclosed systems to a target cell or tissue.EXAMPLESExample 1: DNA-Regulated Enzymes for Amplified Analyte Sensing and Therapy

[0113] Disclosed herein is a biochemical sensor that enables the sensitive, selective, and versatile detection of a wide range of analytes including DNA, non-nucleic acids, and enzymatic activity. These probes include four key and programable components: 1) a binding moiety (DNA aptamer, antibody, covalent reaction group, etc.), 2) an inhibiting moiety (DNAaptamer, small molecule, protein-based inhibitor, etc.), 3) a variable sensing region (DNA, peptide, etc.), and 4) a high-affinity enzyme anchoring moiety (DNA aptamer, antibody, etc.). These four components are attached with the sensing region flanked by the inhibitory and binding moieties, and in this state can bind onto an enzyme of interest through a strong multivalent interaction. The binding of the entire SMDT structure causes the enzyme to go into an inactive state. When an analyte of interest is present, it will interact with the recognition region, cause a structural change in the sensing region, and remove the inhibitory moiety from the enzyme, which then regains its activity. This system has been used so far to detect DNA down to 0.25nM, TATA box binding protein (TBP) down to 5nM, platelet-derived growth factor (PDGF) down to lOnM, C-Myc down to lOnM, manganese ions in the micromolar range, and SARS-CoV-2’s 3CL protease activity. These probes could also be used to modify therapeutic enzymes to allow- for the creation of selectively activatable pro-drugs. Taken together, bivalent aptamer systems form a highly programable and versatile tool for the detection of a wide range of analytes with the capabilities to make advances in therapeutics as well.

[0114] For example, disclosed is a modifiable DNA triblock system that contains a binding aptamer, an inhibitory molecule, and a signaling enzyme. The triblock system can bind to a variable analyte ofinterestto produce a visual signal. It can detect 20 nM ofDNA in 15 minutes by eye. The triblock system can be modified to incorporate a variable peptide region that allow s for the detection of diagnostic protease activities. Analytes that have been successfully detected include: DNA sequences, TATA box binding protein, manganese ions, and 3CL main protease activity from SARS-CoV-2. While the initial focus is on the detection of various analytes, the scope can be broadened to incorporate other signaling enzymes, as well as the inclusion of therapeutically relevant enzymes such as Granzyme B. This will allow for the creation of a selectively activatable therapeutic which responds to analytes in diseased cells to deliver enzymatic treatment.

[0115] The DNA-regulated system offers significant advantages in the areas of signaling and therapy. The inclusion of the variable trigger region allows for the system to be programmable for a wide range of nucleic, non-nucleic. and enzymatic activity biomarkers. This versatility, along with the capabilities of the sensing enzyme for amplification, allow the system to be sensitive to the analyte of interest. The capability of the system to be switched for an activatable therapeutic enzyme, allows for the installation of a disease responsive switch, without the need to modify the native enzyme.

[0116] In this DNA-enzyme system, enzyme activity is controlled using an allosteric DNA switch. Here, an enzyme and its reversible inhibitor are tethered together through a DNA sequence. In this form, the enzyme is inactive due to proximity to its inhibitor. The DNA is designed to be responsive to either nucleic acid or non-nucleic acid targets. Binding of these targets to the DNA changes the DNA conformation as a result of which the enzyme and the inhibitor separate and the enzyme's activity is turned on. Consequently, the system can be used as sensors of these target molecules or as prodrugs that are only activated in diseased tissue containing the target molecules.

[0117] In a prototype system, the enzyme thrombin was used for the detection of both nucleic acid (DNA and RNA), non-nucleic acid targets (including cancer-relevant protein biomarkers and metal ions), and enzymatic activities (disease relevant activity of proteases). The system includes two DNA aptamers linked by a variable DNA or peptide recognition sequence, and the serine protease thrombin. This system allows for the detection of targets isothermally, within a 15-minute period, both by eye and by microplate reader.

[0118] One of the thrombin binding aptamers, aptamer 15 (A15), inhibits thrombin activity, while the other, aptamer 29 (A29), does not inhibit thrombin activity. When these aptamers are linked, they form a bivalent aptamer system that exhibits a binding affinity greater than that of either aptamer alone. The variable region which connects the aptamers can include single stranded DNA (between 10-60 base pairs), a DNA aptamer, a DNAzyme, or protease responsive peptide sequence, and is the variable component responsible for analyte detection. In each detection scenario, a structural transition in the variable region triggers the release of thrombin, which can then act on its substrate, fibrinogen, from which the cleavage generates a turbid solution which can be monitored by eye (visible clots) or by microplate reader (absorbance measurement).

[0119] In the case of nucleic acid targets, the single stranded DNA is designed as the complement to the target DNA, and the target DNA hybridizes with the system’s DNA region. This hybridization is associated with a structural change in the variable DNA region, as the more flexible ssDNA is rendered more rigid upon hybridization, causing the release of free thrombin. When a DNA aptamer for a non-nucleic acid target is integrated into the variable region, the binding of the aptamer with the non-nucleic acid target leads to free thrombin release through structural change.

[0120] For the detection of protease activity, a DNA-Peptide-DNA triblock system was utilized, which incorporates both aptamer 15 and aptamer 29, linked by a variable peptide region. The peptide sequence can be chosen to be the substrate of a particular enzyme ofinterest, namely proteases whose activity has been shown to be linked to various diseases such as cancer, viral infections, heart diseases, and many more. The triblock DNA-Peptide-DNA system is introduced to a thrombin enzyme and interacts the same way as the DNA only system. When a protease of interest is introduced, it can cleave the peptide linker, causing the multivalent binding interaction to similarly disappear. This causes the subsequent disassociation of aptamer 15, and the return of the thrombin activity for signaling. This method provides the framework for a protease activatable enzyme which requires no direct modification of the native enzyme.

[0121] The bivalent aptamer system includes several chemical and biological components:

[0122] 1) Thrombin binding aptamer 15: A DNA aptamer that binds to thrombin with somewhat weak affinity and inhibits thrombin's activity.

[0123] 2) Thrombin binding aptamer 29: A DNA aptamer that binds to thrombin with higher affinity and is used as an anchoring moiety.

[0124] 3) Variable DNA region: A DNA region of variable length (10-60) and sequence is used as a target recognition motif. This region can be tailored to be complementary to the target DNA or be replaced with an aptamer for a specific target, or other DNA structural motif. Upon interaction with the target, this variable DNA region undergoes a structural change that leads to the release of free thrombin.

[0125] 4) Thrombin enzyme: The enzyme is used as a signaling moiety. Upon release from the bivalent aptamer system, thrombin is free to act on its substrate, fibrinogen, cleaving it to form fibrin and causing an increase in turbidity of the analyte solution. This turbidity can be monitored via absorbance or by eye as a measure of enzyme activity and a proxy for analyte detection.

[0126] The bivalent aptamer system can be modified in several ways:

[0127] 1) The linking region can be modified to incorporate different DNA sequences (as previously discussed), peptide sequences, or other targetable biomolecules which can respond to an analyte of interest w ith a structural transition, or structural cleavage.

[0128] 2) The aptamer 29 binding sequence can be replaced with other binding molecules such as antibodies.

[0129] 3) The aptamer 15 sequence can be replaced for other reversible enzymatic inhibitors.

[0130] 4) The thrombin enzyme can be replaced with other enzymes to change the method of signaling (from turbidometry to colorimetry, fluorimetry, etc.) / purpose of the enzyme(could include use for therapeutics, detection, etc.). This would also require the modification of binding and inhibitory- aptamers, which can be adjusted for the enzyme of interest.

[0131] The purpose of the invention is to provide rapid, isothermal, amplified, and accessible detection for nucleic and non-nucleic acid targets. This invention is intended for diagnostics as it can be tailored to detect non-nucleic acid targets for which an aptamer exists. More specifically, cancer-relevant protein biomarkers, metal ions, nucleic acid targets, and protease activity can be detected by eye or plate reader.

[0132] This invention is also poised for therapeutic use. The use of the activatable system could be used to create a pro-drug by use of a therapeutic enzyme (Granzyme B for example), that preferentially gets activated in a diseased cell due to a disease biomarker (DNA or RNA sequence, non-nucleic acids, or elevated protease activity). By responding to these biomarkers, the system would then have the ability to distinguish between healthy and diseased cells and administer the active enzyme preferentially in the diseased cells. This capability to selectively activate and administer a therapeutic is lacking in current therapeutics such as small molecule drugs and protein-based which can cause devastating side-effects when the therapeutic is delivered to healthy cells.

[0133] Other DNA-regulated enzy me biosensors are not capable of detecting non-nucleic acid targets, whereas this system is capable of sensitively detecting non-nucleic acid targets. Additionally, this system provides advantages in the form of rapid results within 15 minutes, the ability to be employed without the use of sophisticated equipment (detection by eye), and the ability to be used isothermally at room temperature. Furthermore, the use of an enzyme as a signaling moiety- allows for the generation of amplified signal even while detecting non- nucleic acid targets or protease activities.

[0134] This bivalent aptamer system addresses the need for rapid, sensitive, isothermal, and accessible detection of various nucleic acid targets in solution. Additionally it is able to detect non-nucleic acid targets with remarkable amplification due to the thrombin’s high catalytic rate. Other systems of this nature are limited by their inability- to detect non-nucleic acid targets while providing amplification, leading to higher limits of detection.

[0135] The system also has implications in creating a new class of selectively activated prodrugs. The capability7for the system to sense for disease biomarkers and then release the active enzy me would allow for more accurate delivery of therapeutic drugs to diseased cells. This could potentially diminish the off-target effects seen in current therapeutics such as cancer drugs or antibiotics.

[0136] This invention has several advantages over extant sensing regimes:

[0137] 1) Amplification: Importantly it provides signal amplification for non-nucleic acid target sensing- one activated enzyme molecule can cleave many substrate molecules rapidly.

[0138] 2) Accessibility: It also allows for isothermal measurement and visual detection — this reduces the need for more sophisticated equipment and improves accessibility.

[0139] 3) Rapidity': This invention can also yield results within 15 minutes.

[0140] 4) Versatility: The DNA recognition region can be tailored for the detection of a wide range of analytes.

[0141] 5) Specificity: The high specificity of DNA aptamers towards their targets grants this system high specificity, with reduced off-target binding.

[0142] 6) Sensitivity': The ability of the enzyme to amplify the signal generated from non- nucleic acid target detection leads to lower detection limits.

[0143] DNA can be pre-folded, which reduces the chance of structural disruptions. For biological and non-biological media, enzy me function can be preserved via sample treatment. Specifically, pH can be controlled through buffer addition, and salts can be spiked into samples.

[0144] This system has been shown to be effective for the detection of both nucleic and non-nucleic acid targets, however, it could be adapted for therapeutic purposes. For example, a similar structure could be realized by chemically attaching a DNA sequence containing a trypsin aptamer and a variable DNA region of a non-nucleic acid target to a try psin enzy me. Upon exposure to the target, the target would be bound by the aptamer and brought into close proximity with trypsin, which could degrade the target.Example 2: Single-Molecule DNA Tweezers Enable Programmable Control of Enzyme Activity via Arbitrary Molecular Cues

[0145] Conformational regulation of enzy mes is commonly achieved through molecular switches embedded within the enzyme or linked externally. These switches have been used in split enzyme systems that reassemble in response to stimuli such as light, pH, temperature, or small molecules. DNA is particularly well suited for building these types of switches because of its predictable hybridization, programmable sequence specificity, and responsiveness to a wide variety of cues including nucleic acids, ions, proteins, and small molecules.

[0146] A compelling example of targeted enzyme control comes from the work of Zhao and colleagues, who developed DNA nanorobots functionalized with nucleolin-targeting aptamers and loaded with thrombin, a serine protease. While systemic delivery' of thrombin would normally induce widespread thrombosis, their system ensured that thrombin was released only in the presence of cancer cells overexpressing nucleolin, resulting in localized clotting and tumor necrosis.

[0147] One notable method of regulation, intrasteric inhibition, was demonstrated by Ghadiri and colleagues using a DNA tether to attach a small-molecule inhibitor to an enzyme. The inhibitor could be displaced through DNA hybridization, restoring enzyme activity. While effective, this and similar strategies generally rely on covalent modification of the enzy me and are limited to nucleic acid inputs. Covalent modifications often involve site-specific mutagenesis or nonspecific chemical conjugation, which can disrupt enzyme structure and function.

[0148] To address these limitations, a study was conducted which designed a singlemolecule, conjugation-free construct that enables allosteric control of enzy me activity7in response to user-defined molecular cues. This construct, called a Single-Molecule DNA Tweezer (SMDT). is minimal in design, easy to synthesize, and does not require complex multi-strand assemblies like DNA origami.

[0149] The SMDT functions by bivalently binding to two distinct epitopes on an enzyme using two DNA aptamers: one that inhibits activity and another that binds at a different site. These aptamers are connected by a tunable DNA sequence that responds to specific molecular inputs. In the absence of input, the structure adopts a “pinched’7conformation that inhibits the enzyme. Upon recognition of a target molecule, the SMDT undergoes a conformational change that disrupts binding and reactivates the enzyme.

[0150] The study chose thrombin as a model enzyme due to its two ell-characterized DNA aptamers. Aptamer Al 5 binds the active site with low affinity and inhibits thrombin, while aptamer A29 binds to a distal site with high affinity' but no inhibitory effect. By linking these aptamers through a programmable DNA sequence, the study created a modular, responsive system that toggles thrombin activity' based on the presence of nucleic acids, proteins, or small molecules.

[0151] This work establishes SMDTs as a simple and versatile platform for non-covalent, allosteric regulation of enzymes. Unlike complex DNA nanostructures, the SMDT is a single molecule that offers programmable, selective, and reversible control over enzyme function, making it broadly applicable to biosensing, diagnostics, and synthetic biology.Materials and Methods

[0152] Buffers Used in this Study: See TABLE 1TABLE 1. Buffers used in this study.

[0153] Materials Used in This Study: Thrombin (Cat. No: 605195-1 OOU), fibrinogen (Cat. No: F3879-1G), bovine serum albumin (Cat. No: A9418-5G), carbonic anhydrase II (Cat. No: C2522-5MG), glucose oxidase (Cat. No: G2133), p-galactosidase (Cat. No: 10105031001), TATA box binding protein (Cat. No: SRP2003-10UG), kanamycin sulfate (Cat. No: 60615-5G), and manganese (II) chloride tetrahydrate (Cat. No: 221279) were purchased from Sigma-Aldrich (St. Louis. MO). c-Myc (Cat. No: 40453) was purchased from BPS Bioscience (San Diego, CA). Platelet-derived growth factor (Cat. No: 220-BB-010) was obtained from Bio-Techne (Minneapolis, MN). Thermo Scientific™ Pierce™ horseradish peroxidase (Cat. No: PI31491) and UltraPure DNase / RNase free distilled water (10-977-023) were purchased from Fisher Scientific (Hampton, NH). RNAse A (Cat. No: 10109169001) was purchased from Roche (distributed by Sigma-Aldrich). All other chemicals were analytical grade.

[0154] General Procedure for Measuring Thrombin Activity Using Fibrinogen Functional Turbidimetric assay (FIFTA): Thrombin activity was assessed using the fibrinogen functional turbidimetric assay (FIFTA). Thrombin was diluted in measurement buffer to 4X the final working concentration. Likewise, the inhibitor or SMDT solution was prepared in folding buffer at 4X the final desired concentration. Thrombin-to-inhibitor molar ratios ranged from 1: 1 to 1: 1000. depending on the specific inhibitor or SMDT employed. Fibrinogen was prepared in measurement buffer at a concentration of 4 mg / mL.

[0155] For each assay, 50 pL of the thrombin solution and 50 pL of the inhibitor solution were combined in a well of a 96-well plate. The mixture was incubated at 25°C for 10 minutes with continuous shaking. Following incubation, 100 pL of the fibrinogen solution was added to each well, bringing the total reaction volume to 200 pL.

[0156] Absorbance at 405 nm was recorded immediately and continuously using either the Agilent BioTek Cytation 5 Imaging Multimode Reader or the Agilent BioTek Synergy Hl Multimode Reader.

[0157] For reactivation experiments, a molecular cue was introduced after the thrombininhibitor incubation but before the addition of fibrinogen. The cue was added in a volume less than 5 pL to minimize its impact on the final reaction volume. The mixture was then incubated for an additional 10-20 minutes at 25°C. Subsequently, the fibrinogen substrate was added, and absorbance monitoring commenced.

[0158] Optimizing Thrombin Concentration: The study first conducted experiments to determine the minimum concentration of thrombin that produces a measurable signal in the FIFTA within 30 minutes. Thrombin was diluted in measurement buffer to 2X the final working concentrations - specifically, 1 nM, 2 nM, 4 nM, and 8 nM. For each condition, 100 pL of the thrombin solution was mixed with 100 pL of fibrinogen solution (4 mg / mL in measurement buffer) in a well of a 96-well plate, yielding a total reaction volume of 200 pL. This resulted in final thrombin concentrations of 0.5 nM, 1 nM, 2 nM, and 4 nM, respectively.

[0159] Absorbance at 405 nm was monitored over time using a plate reader.

[0160] Preparation of Inhibitors / SMDTs for Thrombin Inhibition: All inhibitors / SMDTs were initially dissolved in folding buffer at 4X their final working concentration, where IX corresponds to the final concentration in each well of a 96-well plate. The solutions were then heat-denatured at 95°C for 5 minutes, followed by gradual cooling to 25°C over 30 minutes with continuous shaking at 1500 RPM. This process was performed using the Benchmark Multi-Therm Heating Shaker (Cat. No: H5000-H).

[0161] Inhibition of Thrombin with A15 and A29: Thrombin was diluted in measurement buffer to a concentration of 8 nM. Separately, A15 and A29 solutions were prepared in folding buffer at concentrations of 8 nM, 80 nM, 800 nM, and 8000 nM. Fibrinogen was prepared in measurement buffer at a concentration of 4 mg / mL.

[0162] For each assay condition, 50 pL of the thrombin solution was combined with 50 pL of either Al 5 or A29 solution (at one of the four concentrations) in a well of a 96-well plate. The mixtures were incubated at 25 °C for 10 minutes with continuous shaking. After incubation. 100 pL of the fibrinogen solution was added to each well, resulting in a final reaction volume of 200 pL. This resulted in final concentrations of 2 nM thrombin and 2, 20, 200, or 2000 nM of either Al 5 or A29.

[0163] Absorbance at 405 nm was monitored over time using a plate reader.

[0164] Regulation of Thrombin with Nucleic Acids:

[0165] Inhibition'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. Solutions of individual SMDT variants (RIO to R60) were prepared in folding buffer at concentrations of 0 nM, 8 nM, and 16 nM. Fibrinogen was prepared in measurement buffer at a concentration of 4 mg / mL.

[0166] To evaluate thrombin inhibition, 50 pL of the thrombin solution was combined with 50 pL of an SMDT variant solution (at one of the three concentrations) in a well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking. Following incubation, 100 pL of the fibrinogen solution was added to each well, bringing the final reaction volume to 200 pL. This resulted in final concentrations of 2 nM thrombin and 0, 2, or 4 nM of each R10-R60 SMDT variant.

[0167] Absorbance at 405 nm was monitored over time using a plate reader.

[0168] Reactivation'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. Individual SMDT variants (R10 to R40) were prepared in folding buffer at a concentration of 8 nM. Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0169] For each assay, 50 pL of the thrombin solution was combined with 50 pL of a selected SMDT variant in a well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking. Following this initial incubation, 4 pL of a 1 pM stock solution of the corresponding nucleic acid complement (CIO to C40 and rC15) was added to each well. Each cue was sequence-matched to its respective SMDT variant (e.g.. CIO with R10, C20 with R20. and so on). The cue volume was kept below 5 pL to ensure a negligible effect on the total reaction volume. This resulted in final concentrations of 2nM thrombin, 2 nM SMDT, and 20 nM of the nucleic acid complement. The mixture was then incubated for an additional 10-20 minutes at 25°C to allow reactivation to occur. After this second incubation, 100 pL of the fibrinogen solution was added to each well, bringing the total volume to 200 pL.

[0170] Absorbance at 405 nm was monitored over time using a plate reader.

[0171] Effect of Concentration of C15 on the Reactivation of Thrombin-R15 Complex: Thrombin was diluted in measurement buffer to a concentration of 8 nM, and the R15 SMDT variant was prepared in folding buffer at a concentration of 8 nM. Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0172] To assess the effect of increasing concentrations of the C15 nucleic acid cue on thrombin reactivation, 50 pL of the thrombin solution was mixed with 50 pL of the R15 solution in each well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking to allow for SMDT-mediated inhibition.

[0173] Following this incubation, 4 pL of C15 stock solutions at varying concentrations were added to the wells. The total cue volume was kept below 5 pL in all conditions to minimize its effect on the final reaction volume. The reaction mixtures were incubated for an additional 10-20 minutes at 25°C to allow reactivation to occur. Subsequently, 100 pL of the fibrinogen solution was added to each well, bringing the total reaction volume to 200 pL. The final concentrations in each well were 2 nM thrombin, 2 nM R15, and 0, 0.25, 0.5, 1. 5, 10, or 20 nM C15.

[0174] Absorbance at 405 nm was monitored over time using a plate reader.

[0175] Effect of Mismatches on the Reactivation of Thrombin-R15 Complex: Thrombin was diluted in measurement buffer to a concentration of 8 nM, and the R15 SMDT variant was prepared in folding buffer at a concentration of 8 nM. Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0176] To assess the sequence specificity of C 15-mediated reactivation, 50 pL of thrombin solution was combined with 50 pL of the R15 solution in each well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking to allow for inhibition. Following this incubation, 4 pL of 1 pM stock solutions of C15 variants containing 1, 2, 3, or 4 mismatches, or a scrambled sequence, were added to the wells. The cue volume was kept below 5 pL to minimize any effect on total reaction volume. After cue addition, the mixtures were incubated for an additional 10-20 minutes at 25°C. Subsequently. 100 pL of fibrinogen solution was added to each well, bringing the final volume to 200 pL. Final concentrations in each well were 2 nM thrombin, 2 nM R15, and 20 nM of each Cl 5 variant (including mismatched and scrambled sequences).

[0177] Absorbance at 405 nm was monitored over time using a plate reader.

[0178] Effect of Complement Length on the Reactivation of Thrombin-SMDT Complexes: Thrombin was diluted in measurement buffer to a concentration of 8 nM, and the R15 SMDT variant was prepared in folding buffer at a concentration of 8 nM. Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0179] To investigate the effect of complement length on thrombin reactivation, 50 pL of thrombin solution was combined with 50 pL of the R40 solution in each well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking to allow for SMDT-mediated inhibition. Following this incubation, 4 pL of 1 pM stock solutions of complement strands of varying lengths (CIO, C20, C30, C40, C50, and C60) were added to the wells. The cue volume was kept below 5 pL to ensure minimal impact on the total reaction volume. After cue addition, the samples were incubated for an additional 10-20 minutes at25°C to allow reactivation. Subsequently, 100 pL of fibrinogen solution was added to each well, bringing the final volume to 200 pL. Final concentrations in each well were 2 nM thrombin, 2 nM R40, and 20 nM of each complement strand (C10-C60).

[0180] Absorbance at 405 nm was monitored over time using a plate reader.

[0181] Regulation of Thrombin Activity with Proteins :

[0182] Inhibition'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. Solutions of the SMDT variants R-c-Myc. R-TBP, and R-PDGF were prepared in folding buffer at concentrations of 12 nM (for R-c-Myc) and 8 nM (for R-TBP and R-PDGF). Fibrinogen was prepared in measurement buffer at a concentration of 4 mg / mL.

[0183] To evaluate thrombin inhibition, 50 pL of the thrombin solution was combined with 50 pL of an SMDT solution in a well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking. Following incubation, 100 pL of the fibrinogen solution was added to each well, bringing the final reaction volume to 200 pL. This resulted in final concentrations of 2 nM thrombin and 3 nM R-c-Myc or 2 nM R-TBP / R-PDGF.

[0184] Absorbance at 405 nm was monitored over time using a plate reader.

[0185] Reactivation'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. SMDT variants R-c-Myc, R-TBP, and R-PDGF were prepared in folding buffer at concentrations of 12 nM (R-c-Myc) and 8 nM (R-TBP and R-PDGF). Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0186] For each assay, 50 pL of the thrombin solution was combined with 50 pL of the corresponding SMDT solution in a well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking. Following this initial incubation, 4 pL of a 1 pM stock solution of the corresponding protein cue (c-Myc, TBP, or PDGF) was added to each well. Each cue was matched to its respective SMDT variant. The cue volume was kept below 5 pL to ensure a negligible effect on the total reaction volume. This resulted in final concentrations of 2 nM thrombin, 3 nM R-c-Myc or 2 nM R-TBP / R-PDGF, and 20 nM of the corresponding protein cue. The mixture was then incubated for an additional 10-20 minutes at 25°C to allow reactivation to occur. After this second incubation, 100 pL of the fibrinogen solution was added to each well.

[0187] Absorbance at 405 nm was monitored over time using a plate reader.

[0188] Specificity of Reactivation of Thrombin-R-TBP Complex: Thrombin was diluted in measurement buffer to a concentration of 8 nM, and the R-TBP SMDT variant was prepared in folding buffer at a concentration of 8 nM. Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0189] To assess the specificity of TBP-mediated reactivation, 50 pL of thrombin solution was combined with 50 pL of the R-TBP solution in each well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking to allow for inhibition. Following this incubation, 4 pL of 1 pM stock solutions of various protein cues - TBP, c-Myc, BSA, glucose oxidase (GOx), horseradish peroxidase (HRP), carbonic anhydrase (CA), and 0- galactosidase (0-gal) - were added to the wells. The cue volume was kept below 5 pL to minimize any effect on total reaction volume. After cue addition, the mixtures were incubated for an additional 10-20 minutes at 25°C. Subsequently, 100 pL of fibrinogen solution was added to each well, bringing the final volume to 200 pL. Final concentrations in each well were 2 nM thrombin, 2 nM R-TBP, and 20 nM of each protein cue.

[0190] Absorbance at 405 nm was monitored over time using a plate reader.

[0191] Regulation of Thrombin Activity with Small Molecules:

[0192] Inhibition'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. The SMDT variant R-Kan was prepared in folding buffer at concentrations of 8 nM and 16 nM, respectively. Fibrinogen was prepared in measurement buffer at a concentration of 4 mg / mL.

[0193] To evaluate thrombin inhibition, 50 pL of the thrombin solution was combined with 50 pL of the R-Kan solution in a well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking. Following incubation, 100 pL of the fibrinogen solution was added to each well, bringing the final reaction volume to 200 pL. This resulted in final concentrations of 2 nM thrombin and 2 nM R-Kan.

[0194] Absorbance at 405 nm was monitored over time using a plate reader.

[0195] Reactivation'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. The SMDT variant R-Kan was prepared in folding buffer at concentrations of 8 nM and 16 nM. respectively. Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0196] For each assay, 50 pL of the thrombin solution was combined with 50 pL of the appropriate SMDT solution (R-Kan) in a well of a 96-well plate. The mixtures were incubated at 25°C for 10 minutes with continuous shaking. Following this initial incubation, 4 pL of small molecule stock solution was added to each well — 250 mM kanamycin. The cue volume was kept below 5 pL to ensure a negligible effect on the total reaction volume. This resulted in final concentrations of 2 nM thrombin, 2 nM R-Kan and 5 mM kanamycin. The mixtures were then incubated for an additional 10-20 minutes at 25°C to allow reactivation to occur. After this second incubation, 100 pL of the fibrinogen solution was added to each well.

[0197] Absorbance at 405 nm was monitored over time using a plate reader.

[0198] Regulation of Thrombin Activity with Mn2+:

[0199] Inhibition'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. The R-Sub SMDT variant was prepared in folding buffer at a concentration of 16 nM. Fibrinogen was prepared in measurement buffer at a concentration of 4 mg / mL.

[0200] To evaluate thrombin inhibition, 50 pL of the thrombin solution was combined with 50 pL of the R-Sub solution in a well of a 96-well plate. The mixtures were incubated at 25 °C for 10 min with continuous shaking. Following incubation. 100 pL of the fibrinogen solution was added to each well, bringing the final reaction volume to 200 pL. This resulted in final concentrations of 2 nM thrombin and 4 nM R- Sub.

[0201] Absorbance at 405 nm w as monitored over time using a plate reader.

[0202] Reactivation'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. The R-Sub SMDT variant was prepared in folding buffer at a concentration of 16 nM. Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0203] 200 pL of R-Sub + E was combined with 2 pL of 10 mM MnCh stock (i.e., treatment of R-Sub+E with 100 pM Mn2+). This mixture was incubated at 25 °C for 8 h with continuous shaking. In all figures involving Mn2+. we report this Mn2+concentration, as this is the condition under which Mn2+-dependent cleavage occurs. Following this initial incubation, 50 pL of R-Sub + E + Mn2+solution was combined with 50 pL of the thrombin solution in a well of a 96-well plate. The mixtures were incubated at 25 °C for 10 min with continuous shaking. After this second incubation, 100 pL of the fibrinogen solution was added to each well. This resulted in final concentrations of 2 nM thrombin. 4 nM R-Sub + E, and 25 pM Mn21.

[0204] Absorbance at 405 nm was monitored over time using a plate reader.

[0205] Mn2+controls '. It was observed that Mn2+alone increases the activity of thrombin. To account for this effect, 100 pM Mn2+(from a 10 mM stock) was also incubated with pots containing only thrombin and fibrinogen (in the absence of R-Sub). Additionally. R-Sub and E were treated in the same manner as additional controls.

[0206] Regulation of Thrombin Activity with RNase:

[0207] Inhibition'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. The R-RNase SMDT variant was prepared in folding buffer at a concentration of 256 nM. Fibrinogen w as prepared in measurement buffer at a concentration of 4 mg / mL.

[0208] To evaluate thrombin inhibition, 50 pL of the thrombin solution was combined with 50 pL of the R-RNase solution in a w ell of a 96-well plate. The mixtures w ere incubated at 25 °C for 10 minutes with continuous shaking. Following incubation, 100 pL of the fibrinogen solution was added to each well, bringing the final reaction volume to 200 pL. This resulted infinal concentrations of 2 nM thrombin and 64 nM R-RNase. This resulted in final concentrations of 2 nM thrombin and 4 nM R-Mn2+.

[0209] Absorbance at 405 nm was monitored over time using a plate reader.

[0210] Reactivation'. Thrombin was diluted in measurement buffer to a concentration of 8 nM. The R-RNase SMDT variant was prepared in folding buffer at a concentration of 256 nM. Fibrinogen was prepared in measurement buffer at 4 mg / mL.

[0211] For each assay. 50 pL of the thrombin solution was combined with 50 pL of the R- RNase solution in a well of a 96-well plate. The mixtures were incubated at 25 °C for 10 min with continuous shaking. Following this initial incubation, 4 pL RNase stock solution (variable concentration) was added to each well. The cue volume was kept below 5 pL to ensure a negligible effect on the total reaction volume. This resulted in final concentrations of 2 nM thrombin, 64 nM R-RNase, and 100, 1000 or 2000 nM RNase. The mixtures were then incubated for an additional 10-20 min at 25 °C to allow reactivation to occur. After this second incubation, 100 pL of the fibrinogen solution was added to each well, bringing the total volume to 200 pL.

[0212] Absorbance at 405 nm was monitored over time using a plate reader.

[0213] Electrophoretic Mobility Shift Assays (EMSA): Native PAGE gel was used to study the interactions between thrombin, SMDTs, and molecular cues. Samples were prepared by mixing thrombin, SMDTs, and molecular cues at appropriate ratios in folding buffer. 20 pL of each sample was mixed with 4 pL of glycerol and loaded onto a 4-15% acrylamide gel (BioRad, 4-15% Mmi-PROTEAN TGX Precast Protein Gels, 10-well, Cat. No. 4561084). The gel was run for 60 minutes at 100V and at room temperature. To create the running buffer, 10X Tris-Borate-EDTA (TBE Buffer, Thermo Fisher, Cat. No. BP13334) was diluted to obtain a IX TBE Buffer. The DNA and thrombin-DNA complex bands were visualized by GelRed dye (Biotium, Inc., Hayward, CA, USA cat# NC9594719) according to the manufacturer's protocol. Images were captured using the ChemiDoc MP Imaging System (Bio-Rad, USA). Subsequently, the protein bands of same gel were stained with Pierce™ Silver Stain Kit (Thermo Fisher, Cat. No. 24612), according to the manufacturer's protocol, and reimaged. The EZ-Run™ Prestained Rec Protein Ladder (Fisher Scientific, Cat. No: BP3603500), Tracklt™ Ultra Low Range DNA Ladder (Invitrogen, Cat. No: 10-488-023), and Gel Loading solution Type I 6X (Sigma Aldrich, Cat. No: G7654-5ML) were utilized in gel electrophoresis to facilitate molecular weight determination and sample tracking.

[0214] Transmission Electron Microscopy (TEM): Four hundred mesh continuous carbon grids with copper support (Electron Microscopy Sciences) were glow discharged on aPELCO easiGlow for 40 seconds at a current of 20 mA (Ted Pella, Inc). 4 pL of sample was applied to the grid at a concentration of 5 pM for the thrombin-R15 sample and 2.5 pM for thrombin only sample. Samples were blotted by hand on a filter paper after a 1 minute wait time and promptly washed in five 20 pL droplets of 2% aqueous Uranyl Acetate at pH 4.5 for 5 seconds each. After a 30 second wait time, the grid was again blotted by hand on a filter paper and air dried in a fume hood. Samples were loaded into a TFS Tecnai Spirit TEM operating at a high tension of 80 kV. Images were taken at a nominal magnification of 87,000x on a sidemount AMT Advantage HR CCD Ik x Ik camera (9.31 A / pixel). Collected micrographs were uploaded to cryoSPARC v4.5.1 for CTF estimation, particle picking, and 2D class averaging. Fifty classes were used to align 3,414 particle picks for the thrombin-R15 dataset and 9,931 particle picks for the thrombin only dataset.

[0215] Data Analysis: All reported data represent the average of at least three replicate measurements, unless otherwise noted. Error bars indicate the standard deviation of these measurements.

[0216] To calculate activity (%) for a sample, the absorbance of fibrinogen alone (AF) is first subtracted from both the sample (As) and the thrombin control (AT) to correct for background. The background-corrected sample absorbance is then normalized to the background-corrected thrombin absorbance, which is set to 100%. This allows for consistent comparison of residual thrombin activity across different conditions.A — A Activity (%) = — - - x 100%A Ap

[0217] Thrombin reactivation is expressed as AS / AF, where Asis the absorbance of the sample and AFis the absorbance of fibrinogen alone (i.e., without thrombin) at the same time point. Since fully inhibited thrombin produces a signal similar to fibrinogen alone, an AS / AFvalue greater than 1 indicates a fold increase in signal upon addition of a reactivating molecular cue.

[0218] Time-dependent changes in absorbance are presented as At / A0, where Atis the absorbance at time t, andis the absorbance at the initial time point. This ratio illustrates how the signal evolves over time relative to its starting value.Results and Discussion

[0219] Design of Single-Molecule DNA Tweezers: The SMDT structure (FIG. 1) includes a central recognition moiety that detects the target molecular cue, flanked by twodistinct aptamers that bind to different sites on the enzy me. One of these aptamers acts as an inhibitor, keeping the enzyme in an ■■off-state'’ when the SMDT is bound. When the target molecular cue is introduced, the SMDT undergoes structural rearrangements, causing the inhibitory aptamer to detach from the enzyme. This removal of inhibition restores the enzy me’s activity, effectively syvitching it to an ‘"on-state.” As a result, the SMDT functions as a heterodimeric, conditional binder, with its enzyme-binding ability controlled by external molecular cues.

[0220] Thrombin serves as an ideal enzyme for demonstrating this design, as it has two well-characterized binding sites that interact with distinct aptamers (FIG. 2A). A 15-nt DNA aptamer (Al 5) binds electrostatically to thrombin’s fibrinogen recognition site, blocking its ability to bind and cleave fibrinogen (FIG. 4, FIG. 5). In contrast, a 29-nt DNA aptamer (A29) binds to the heparin-binding site through hydrophobic interactions but does not interfere with thrombin’s activity (FIG. 4). By leveraging these distinct binding interactions, the study designed an SMDT that links these aptamers via a variable single-stranded DNA (ssDNA) sequence (TABLE 2, TABLE 3). This linking region contains a recognition moiety (R), enabling the SMDT to respond to specific molecular cues and modulate thrombin activity in a controlled manner. To minimize potential steric hindrance, the linking region also incorporates a 3 -nt spacer between R and each aptamer.TABLE 2. Nucleic acid-responsive SMDT sequences and complements used in this study. Bold sequences correspond to Al 5 and A29. Underlined sequences correspond to the recognition moiety7.TABLE 3. Non-nucleic acid-responsive SMDT sequences used in this study. Bold sequences correspond to Al 5 and A29. Underlined sequences correspond to the recognition moiety.

[0221] The cooperative binding of A15 and A29 within the SMDT markedly enhances its binding affinity for thrombin compared to either aptamer alone. This synergistic effect was validated using electrophoretic mobility shift assays (EMSAs), as shown in FIG. 5, FIG. 6, and FIG. 7. This increased affinity arises from multivalency, as both aptamers simultaneouslyengage thrombin, stabilizing the interaction. These results are consistent with previous reports showing that combining A 15 and A29 into a single molecular entity creates a stronger thrombin binder and inhibitor. Consequently, the SMDTs developed in this study can inhibit thrombin at up to 1000-fold lower concentrations compared to Al 5 (FIGS. 2B-2C). More specifically, SMDTs inhibited 2 nM thrombin at SMDT concentrations below 10 nM, whereas A15 alone required concentrations exceeding 2 pM to achieve comparable inhibition (FIG. 2D).

[0222] The length of the linking region is a critical factor in determining the extent of cooperative binding. If the linker is too short, it may hinder the simultaneous binding of both aptamers to thrombin, reducing the cooperative effect. Conversely, if the linker is too long, the aptamers may bind independently rather than cooperatively, weakening overall binding strength. Thus, optimizing the linker length is essential for maximizing the SMDT’s inhibitory efficiency.

[0223] To inform the linker design, the study considered the spatial arrangement of thrombin’s binding sites. The distance between the two exosites of thrombin has been reported to be 3.4-4.4 nm. Using the PDB structure of human thrombin from PDB ID: 5EW1, the study measured this distance to be 3.6 nm. which falls within the reported range. However, since the SMDT must extend across the enzyme’s surface rather than spanning a linear distance through the enzyme, the study estimated that the linker must be longer than ~8.5 nm to effectively bridge both binding sites.

[0224] Therefore, the study designed a series of SMDTs with variable linker lengths by adjusting the length of R from 10 to 60 nt. Given that the length of ssDNA is reported to be ~0.6 nm per nt depending on ionic strength, the total estimated linker length ranged from 9.6 nm (lower bound) to 39.6 nm (upper bound). These SMDTs, referred to as R10 through R60, allowed the study to systematically assess the impact of linker length on thrombin inhibition.

[0225] To evaluate the effect of linker length, the study incubated 2 nM and 4 nM of each SMDT with 2 nM thrombin and measured thrombin’s ability to convert fibrinogen to fibrin (FIG. 2D). As fibrinogen is cleaved, the solution becomes turbid, and this reaction can be monitored by measuring the absorbance at 405 nm. All SMDTs functioned as effective thrombin inhibitors, demonstrating greater inhibition than Al 5 alone. However, the extent of inhibition varied with linker length, following the trend: SMDTs with the shortest recognition moiety (R10) exhibited weaker inhibition, likely due to insufficient linker length preventing simultaneous aptamer binding. The strongest inhibition was observed with R15, suggesting that this linker length provides optimal spatial positioning of the aptamers for cooperativebinding. Beyond this point, as the length of R increased, inhibition efficiency gradually declined, likely due to excessive linker length diminishing cooperative effects.

[0226] These results led to the hypothesis that molecular cues capable of altering the SMDT structure and disrupting cooperative binding would facilitate thrombin reactivation.

[0227] Programmable Regulation through the Binding of Complementary Nucleic Acids: SMDTs regulate thrombin activity by transitioning between two conformational states. In the “pinched” conformation, the SMDT binds to thrombin, keeping it in an “off-state” and inhibiting its activity. Upon binding to a specific molecular cue, the SMDT shifts to the “released” conformation, detaching from thrombin and restoring its activity (“on-state”).

[0228] This transition can be triggered by nucleic acid inputs, such as DNA or RNA, that are complementary to R (FIG. 8). Single-stranded DNA (ssDNA) is highly flexible, with a persistence length of less than ~2 nm, whereas double-stranded DNA (dsDNA) is significantly more rigid, with a persistence length of ~50 nm. Consequently, hybridization with complementary nucleic acids via Watson-Crick-Franklin base pairing increases the rigidity of the linking region between Al 5 and A29, disrupting the optimal spacing between the aptamers and preventing them from simultaneously binding to thrombin’s two exosites.

[0229] When the concentrations of both thrombin and SMDTs are significantly lower than the dissociation constant (Ka) of A15, the loss of cooperative binding between A15 and A29 leads to the dissociation of Al 5 from thrombin, thereby restoring its enzymatic activity. This structural change drives the SMDT into the “released” state.

[0230] FIG. 8 illustrates the reactivation of 2 nM thrombin following incubation with SMDTs R10-R40 at a 1:1 ratio and subsequent treatment with 20 nM nucleic acid sequences complementary to R. Thrombin regulation was observed across all tested SMDTs exposed to their respective complementary DNA sequences (C). R50 and R60, whose “pinched” state exhibited poor thrombin inhibition at 1 : 1 thrombin-to-SMDT ratios could be toggled between “pinched” and “released” states by combining thrombin, SMDTs, and corresponding complementary' strands, provided a higher SMDT concentration was used (FIG. 9).

[0231] To further investigate the factors influencing reactivation, the study focused on SMDT R15. which demonstrated the strong thrombin inhibition at a 1 : 1 ratio. The study assessed the impact of complementary strand (Cl 5) concentration on reactivation and found that thrombin activity was restored in a concentration-dependent manner, with as little as 0.25 nM Cl 5 being sufficient to induce reactivation (FIG. 8C). Additionally, replacing the DNA cue with a complementary RNA sequence (rC15) also successfully restored thrombin activity, demonstrating that the regulatory mechanism is driven by hybridization (FIG. 10).

[0232] The specificity of SMDTs to molecular cues was also evaluated (FIG. 8D). When a scrambled DNA sequence that did not complement the recognition moiety of R15 was introduced, no thrombin reactivation was observed, confirming the sequence-dependent nature of activation. To further investigate the role of sequence complementarity, the study tested complementary' sequences containing 1, 2, 3, or 4 base mismatches (FIG. 8D). Notably, SMDTs exhibited high sensitivity to even a single base mismatch, with reactivation efficiency decreasing progressively as the number of mismatches increased.

[0233] To evaluate the influence of rigidity, the study examined a medium-length SMDT (R40) with complementary DNA inputs of varying lengths (FIGS. 8E-8F). Shorter input sequences (C10-C30) failed to reactivate thrombin because the unhybridized ssDNA bases provided sufficient flexibility to maintain the cooperative binding of SMDT to thrombin. In contrast, longer sequences (C40-C60) successfully restored thrombin activity, due to increased rigidity that disrupted cooperative binding. These findings highlight the importance of a structural rigidity threshold for effective reactivation, as shorter inputs lack the stiffness needed to displace the SMDT-thrombin interaction.

[0234] Collectively, these results demonstrate that enzyme activity can be precisely regulated using nucleic acids as molecular cues through SMDTs, with concentration, sequence specificity', and changes in structural rigidity' serving as key determinants of control. These findings underscore the critical role of nucleic acid design in modulating enzyme activity and highlight its potential for programmable biomolecular regulation.

[0235] Programmable Regulation through Protein and Small-Molecule Binding: The study next explored how SMDTs could be adapted to respond to molecular cues beyond complementary' nucleic acids. Specifically, it was hypothesized that if the R domain were designed as a DNA sequence that binds a target molecule with high specificity (e.g., an aptamer sequence), the binding event could induce a conformational change in the SMDT. If this structural change were sufficient to disrupt the cooperative interaction between A15 and A29, it could trigger the release of active thrombin.

[0236] To test this concept, the study selected three proteins as molecular inputs: cellular myelocytomatosis (c-Myc), platelet-derived growth factor (PDGF), and TATA-binding protein (TBP). The study incorporated DNA sequences known to bind each of these targets into the linker region, generating three constructs: R-c-Myc, R-PDGF, and R-TBP. FIG. 11A illustrates the predicted structural transitions of each SMDT upon target binding, based on previous literature.

[0237] For all three proteins, it was hypothesized that target binding to the SMDT would reduce the distance between Al 5 and A29 below the minimum required for cooperative binding, thereby restoring thrombin activity. Initial tests with R-c-Myc, in which the R domain included the c-Myc-binding sequence, showed minimal thrombin inhibition, even at a 1 :4 thrombin-to-SMDT ratio (FIG. 12A). The study attributed this to the R domain being too short to span the distance between A15 and A29 (estimated linker length: -2 nm). To extend the linker, the study added 3-nt spacers to both ends of the c-Myc recognition sequence (estimated linker length: ~4.6 nm), but inhibition remained weak (FIG. 12B), consistent with the calculated minimum requirement of ~8.5 nm. When the spacers were further extended to 6 nt on each end (estimated linker length: -11.8 nm), robust thrombin inhibition was observed at a 1:4 ratio (FIG. 13), with measurable inhibition even at 1 : 1.5 (FIG. 11B). Upon addition of 25 nM c-Myc protein to a solution of 2 nM thrombin and 3 nM R-c-Myc, thrombin activity was restored, confirming successful reactivation via protein binding (FIG. 11B).

[0238] R-PDGF also demonstrated effective thrombin inhibition at a 1 : 1 ratio and could be reactivated by adding 20 nM PDGF (FIG. 11C). Similarly, R-TBP inhibited thrombin efficiently at a 1: 1 ratio and was reactivated with 20 nM TBP (FIG. 11D).

[0239] R-TBP was selected for further assessment as it displays the highest level of reactivation. Thus, the study further investigated the activation threshold of the TBP-regulated tweezer in terms of concentration. As shown in FIG. HE, as the TBP concentration increases from 1 nM to 20 nM, thrombin reactivation increases in a concentration dependent manner, highlighting the tunability of this system.

[0240] The tweezer system also displayed high molecular specificity. The R-TBP construct was exposed to a panel of unrelated proteins, including c-Myc, bovine serum albumin, carbonic anhydrase, horseradish peroxidase, glucose oxidase, and |3-galactosidase (FIG. HF). None of these proteins triggered reactivation, underscoring the specificity of the design.

[0241] To further expand the scope of this approach beyond protein inputs, the study incorporated a DNA aptamer that binds the small molecule kanamycin into the R domain, generating R-Kan. This construct successfully inhibited 2 nM thrombin at a 1: 1 thrombin-to- SMDT ratio, and thrombin activity was restored upon the addition of 5 mM kanamycin (FIG. 11G)

[0242] Collectively, these results demonstrate that SMDTs can be broadly programmed to respond to diverse classes of molecular inputs - including both proteins and small molecules - establishing them as a highly versatile platform for targeted biomolecular activation.

[0243] Programmable Regulation through Chemical Activity: Thus far, the study has demonstrated that SMDT activation can be driven by molecular cues based on their abundance. However, SMDTs can also be adapted to respond to cues from chemical activity. It was hypothesized that if the linking region were cleaved in response to the activity of a molecular cue, it would trigger thrombin release by disrupting the cooperative multivalent binding of the aptamers.

[0244] To test this, the study designed an SMDT in which the linking region incorporates the substrate strand for a well-characterized Mn2+-activated DNAzyme. This substrate strand contains a single RNA adenine base (rA) flanked by DNA sequences that hybridize with the enzyme strand of the DNAzyme (FIG. 14A). This SMDT, termed R-Sub + E , was mixed with thrombin at a 1 :2 ratio, resulting in inhibition of thrombin activity (FIG. 14B). As shown in FIG. 14C, 100 pM Mn2+was sufficient to trigger thrombin reactivation.

[0245] The study further designed an SMDT that responds to RNAse A activity, an enzyme known to catalytically degrade RNA (FIG. 14D). In this case, R included a 15-nt rU sequence, chosen based on previous experiments showing that this length provided optimal thrombin inhibition. Similar to the DNAzyme mechanism, cleavage of the linking region by RNase A led to thrombin reactivation, as illustrated in FIG. 14E.

[0246] RNase A was selected for further assessment due to its ability to activate thrombin at nanomolar concentrations. Thrombin activation increased in a concentration-dependent manner with RNase A, demonstrating the tunability of the system (FIG. 14E). Additionally, when the recognition moiety was replaced with a 15-nt DNA sequence, no activation was observed (FIG. 14F).

[0247] These results demonstrate that SMDTs can be designed to respond not only to molecular abundance but also to molecular activity, expanding their potential applications in biosensing and controlled biomolecular activation.Additional Results and Discussion

[0248] Determining the Optimal DNA Linker Length for Effective Thrombin Inhibition by SMDTs: To estimate the minimum linker length required for cooperative binding of aptamers Al 5 and A29 to thrombin, the study applied a simplified geometric model.

[0249] Using the crystal structure of human thrombin (PDB ID: 5EW1), in which both A15 and A29 are bound simultaneously, the study first characterized thrombin’s geometry'. Given its globular shape, the study approximated thrombin as a sphere with a diameter of 4.5 nm. From this structure, the study measured the linear distance between the 3’ end of Al 5 and the 5' end of A29 to be approximately 6.5 nm (FIG. 15).

[0250] To model the DNA linker, the study treated single-stranded DNA (ssDNA) as a flexible cylinder with a diameter of ~1 nm, based on the known 2 nm diameter of doublestranded DNA (dsDNA). Because A15 and A29 bind on nearly opposite sides of the thrombin surface, it was assumed that the shortest path the linker could take - while remaining closely wrapped around thrombin - would he along a spherical shell, denoted as S, concentric with thrombin (FIG. 16).

[0251] The radius of this shell (rs) is the sum of the thrombin radius (rT) and the ssDNA radius (rssDNA): rs = rT+ rssDNA

[0252] The study estimated the minimum contour length of the linker by summing three components:

[0253] i) The straight-line distance from the 3’ end of Al 5 to the point where its tangent meets the shell S.

[0254] ii) The analogous straight-line distance from the 5’ end of A29 to its respective tangent point on S.

[0255] iii) The arc length between the two tangent points along the spherical shell S.

[0256] Together, these components define the minimum path the ssDNA linker must follow while maintaining close contact with the thrombin surface. This total contour length was calculated to be 8.54 nm. Given that ssDNA has an approximate contour length of 0.6 nm per nucleotide, this corresponds to a minimum linker length of 14 nucleotides.

[0257] When designing SMDTs to be responsive to nucleic acid cues, the study introduced 3-nt T spacers on each side of the recognition moiety (R) to provide flexibility and minimize steric hindrance. Consequently, the minimum effective length of the recognition sequence itself is reduced to 14 - 3 * 2 = 8 nucleotides. However, such an 8-nt sequence typically lacks sufficient melting temperature to stably hybridize to its complementary strand under physiological conditions.

[0258] Based on these considerations, the study designed a series of SMDTs with variable linker lengths by adjusting the length of the recognition region R from 10 to 60 nucleotides, corresponding to physical lengths of approximately 9.6 nm to 39.6 nm. These values account for the 3-nt T spacers on either side of the recognition region, which remain constant across designs.

[0259] Based on the linker length calculations, it was hypothesized that all of the designed SMDTs would support cooperative binding of Al 5 and A29 to thrombin, resulting in potentinhibition at concentrations lower than those required for Al 5 alone. Experimental results support this hypothesis (FIG. 2).

[0260] Optimizing Thrombin Concentration: The study first investigated what the minimum concentration of thrombin would give reasonable signal within a short (<30 min) time frame. To this end, different concentrations of thrombin (4, 2, 1, and 0.5 nM) were assayed via FIFTA. It was observed that both 2 nM and 4 nM thrombin reached a saturation point within 15 min. 2 nM thrombin was thus selected as optimal in the interest of saving material.

[0261] Effect of A15 and A29 on T Activity: Al 5 binds to T with relatively poor affinity, (rel) requiring increased concentrations to fully inhibit thrombin activity'. To confirm this weak inhibitory' effect, T (2 nM) was incubated with increasing concentrations of Al 5 (0, 2, 20, 200, and 2000 nM), and clotting activity was monitored over 15 min. Significant inhibition of thrombin activity' was observed at very high A15 concentrations (>200 nM), indicating that A15 binds thrombin with relatively low affinity. Concentrations below 20 nM showed minimal to no inhibitory' effect on thrombin activity'.

[0262] A29 binds to the heparin-binding site of thrombin rather than the fibrinogen-binding site and therefore does not interfere with thrombin’s fibrinolytic catalytic activity'. To confirm this behavior, 2 nM thrombin was incubated with increasing concentrations of A29 (0, 2, 20, 200, and 2000 nM). Unlike A15, A29 did not inhibit thrombin activity' at any tested concentration, indicating a lack of inhibitory interaction.

[0263] Comparison of Binding Interactions Between T and A15, A29, and SMDT Variants: To evaluate the relative T-binding efficiency of different constructs, native gel electrophoresis was performed using A15, A29, and R15, each at 400 nM, both in the presence and absence of T (400 nM). Upon addition of T, a new. slower-migrating band appeared specifically in the R15 lane, indicative of a stable T-R15 complex. This observation was further corroborated by silver staining, which revealed the same mobility shift. In contrast, no such band shift was observed for Al 5 or A29 under identical conditions. These results demonstrate that R15 has markedly higher T-binding efficiency compared to monovalent A15 or A29.

[0264] The study next examined the binding interactions betw een T and a series of SMDT (R) variants to confirm that all constructs are capable of binding. Native gel electrophoresis was performed using R10, R15, R20, R30, R40, R50, and R60, each at 200 nM, both in the presence and absence of thrombin (200 nM). As show n in FIG. 6, each construct exhibited a distinct migration pattern in the absence of thrombin. Upon thrombin addition, all variants displayed a new, slower-migrating band in the GelRed™ channel, indicating complex formation. These results demonstrate that all SMDT constructs are capable of binding to T.This conclusion was further supported by silver staining, which showed co-localization of the protein signal with the shifted DNA bands, confirming the formation of stable T-SMDT complexes.

[0265] Comparison of Binding Affinity Between T and A29 Versus R50: Electrophoretic mobility shift assays (EMSAs) were used to assess how much more strongly SMDT constructs bind to T compared to monovalent aptamers. A29 was selected as the monovalent comparator because A15 did not produce clearly stainable bands in the gel, making it unsuitable for quantitative analysis. Although R15 was identified as the most effective inhibitor of T activity, R50 was chosen for EMSA-based binding affinity estimation because it consistently produced detectable T-SMDT complex bands even at low T concentrations, whereas R15 did not. It is important to note that the binding affinities derived from EMSA reflect relative differences within the gel matrix and may not precisely correspond to solutionphase affinities due to differences in buffer conditions. Nevertheless, EMSAs provide a useful approximation of binding strength, allowing estimation of how much more effectively SMDTs interact with T compared to monovalent constructs.

[0266] To investigate the binding interaction between A29 and thrombin, native gel electrophoresis was performed with increasing thrombin concentrations (200-1600 nM) in the presence of 400 nM A29. To validate these findings, the gel was sequentially stained with GelRed (FIG. 7A) to visualize DNA bands and with silver stain (FIG. 7B) to detect protein. As shown in FIGS. 7A-7B, a distinct mobility shift was observed in lanes 3 through 8, indicating complex formation between A29 and thrombin. The intensity of the shifted bands increased with rising thrombin concentrations, suggesting a concentration-dependent interaction. No shift was observ ed for A29 alone (lane 2) or thrombin alone (lane 9), confirming the specificity of the binding. The colocalization of DNA and protein signals in the shifted regions confirms the formation of a specific and stable aptamer-thrombin complex. These results demonstrate that A29 binds thrombin in a concentration-dependent manner under native conditions.

[0267] Gel with Various Thrombin Concentrations and Fixed R50 Concentration: To assess the binding interaction between R50 and thrombin, native gel electrophoresis was performed using a fixed concentration of R50 (200 nM) with increasing concentrations of thrombin (25-800 nM). As shown in FIG. 17A, stained with GelRed, a progressive mobility shift was observed in lanes 3 through 8, corresponding to the increasing thrombin concentrations. This shift indicates the formation of R50-thrombin complexes in aconcentration-dependent manner. Lane 2, containing R50 alone, shows no shifted band, while lane 9 (thrombin alone) lacks DNA signal, confirming binding specificity’.

[0268] To validate the presence of protein in the shifted complexes, the same gel was subsequently stained with silver stain (FIG. 17B). The silver staining confirmed the presence of thrombin co-migrating with the DNA bands, further supporting specific complex formation. These results collectively demonstrate that R50 interacts specifically and strongly with thrombin in a concentration-dependent manner under native conditions.

[0269] Reactivation of T-SMDT Complexes R50 and R60 with Complementary Nucleic Acid Sequences: To confirm that the regulatory' mechanism of T-SMDT complexes is driven by hybridization, thrombin (2 nM) was incubated with R50 and R60 constructs followed by the addition of complementary nucleic acid sequences (C50 and C60). The introduction of complementary strands successfully reactivated thrombin, restoring activity to levels comparable to the thrombin control. This confirms that the regulatory' mechanism is mediated by sequence-specific hybridization, effectively reversing the inhibitory' state of the SMDT complexes.

[0270] Reactivation of T-SMDT Complex with RNA: To confirm that the regulatory mechanism of SMDTs is driven by hybridization, a 2 nM thrombin-R15 complex was exposed to rC15, an RNA sequence complementary' to the recognition domain. The addition of rC15 successfully reactivated thrombin, mirroring the effect observed with C15. This demonstrates that the regulatory mechanism is indeed mediated by sequence-specific hybridization.

[0271] Programmable Regulation through Protein and Small-Molecule Binding:

[0272] Structural Optimization of c-Myc SMDT R Domain'. See FIG. 12.

[0273] Effect of Increasing R Domain Length on SMDT-c-Myc Thrombin Inhibition'. To assess the effect of nucleotide spacer length on the inhibitory’ efficiency of the R-cMyc construct, the spacer length was varied from 0 nt to 3 nt, and thrombin inhibition was evaluated using a FIFTA assay.

[0274] As shown in FIG. 13, thrombin alone (T) displayed full cataly tic activity', while the fibrinogen-only control (F) exhibited no significant activity. Increasing the spacer length from 0 nt (R-cMyc 1) to 3 nt (R-cMyc 2) enhanced thrombin inhibition, as indicated by a notable reduction in thrombin activity in the R-cMyc 2 construct. The R-cMyc 1 construct showed comparatively weaker inhibition, suggesting that a longer spacer length facilitates more effective aptamer binding by reducing spatial constraints.

[0275] These results indicate that spacer length is a critical parameter in modulating thrombin inhibition by SMDT-cMyc constructs, with longer spacers providing stronger inhibition through improved aptamer binding affinity.

[0276] Allosteric Regulation through Chemical Activity:

[0277] Gel Confirms DNAzyme Cleavage Activity is Activated By An2Mn2+-activated DNAzyme cleavage activity was confirmed by using native PAGE. The hybridization of 100 nM of the catalytic enzyme strand (E) and the substrate strand (R-Sub) led to the formation of anew DNAzyme band (highlighted within the box). The intensity of this band is significantly diminished when the E-R-Sub complex is treated with 100 pM Mn2+. This is because Mn2+causes catalytic cleavage of R-Sub. (FIG. 18).

[0278] To validate the inhibition of thrombin with R-RNase and its reactivation by RNase A treatment, native gel electrophoresis was performed as shown in FIG. 19. Formation of a complex between R-RNase and thrombin (T) gave rise to a new- band (highlighted in red box). Upon treatment with increasing concentrations of RNase A, the intensity of the complex band progressively decreased, accompanied by the appearance of lower molecular weight bands corresponding to cleaved fragments of R-RNase. These results corroborate the FIFTA data and confirm that RNase A-mediated cleavage effectively reactivates thrombin by disrupting the RNA-based dimer structure.

[0279] These results corroborate the FIFTA data and confirm that RNase A-mediated cleavage effectively reactivates thrombin by disrupting the RNA-based dimer structure.Example 3: Engineering Peptide-DNA Hybrid Probes for Visual Detection of Protease Activity

[0280] Proteases are enzymes that play a vital role in normal biochemical functions. However, in many diseases, the activity of these enzymes becomes dysregulated. Current detection methods for protease activity are limited. These highlights the urgent need for advanced diagnostic tools to better detect and manage these diseases.

[0281] A study w as conducted to develop a rapid, visual platform for protease sensing. A structure of the probe is shown in FIG. 20. The probe was tested for protease activity sensing through thrombin generated turbidity. Thrombin’s activity is inhibited by the DNA-peptide- DNA triblock ligand. When a specific protease cleaves the peptide (protease recognition sequence), it triggers the dissociation of Al 5 (inhibitors DNA) from thrombin, thereby activating it. The active thrombin then converts fibrinogen into fibrin, producing a cloudy signal detectable by eye or a plate reader. A schematic is shown in FIG. 21.

[0282] The triblock ligand was synthesized using solid-phase DNA synthesis and conjugation techniques. A schematic is shown in FIG. 22. A29 with a DBCO group was synthesized on a solid support, followed by peptide attachment via DBCO-azide click chemistry and an azide linker using NHS ester chemistry, incorporating Al 5 with a dye. After purification and deprotection, UV-Vis and gel electrophoresis was used to verify successful synthesis. These results are shown in FIG. 23.

[0283] The tnblock ligand was next tested as a protease-responsive switch. The DNA- peptide-DNA (triblock) exhibits higher binding affinity compared to individual Al 5, A29, and their mixture (Al 5 + A29). In the presence of 3CL protease, the peptide within the triblock is cleaved, reactivating thrombin, which generates a signal with substrate fibrinogen. These results are shown in FIGS. 24A-24B.

[0284] Finally, the probe was tested for sensitivity and selectivity. The probe demonstrates the ability to detect active 3 CL protease down to ~pM concentrations with high selectivity, representing a significant improvement over existing commercial probes. These results are shown in FIG. 25.Materials and Methods

[0285] Buffers used in this study: See TABLE 4.TABLE 4. Buffers used in this study.

[0286] Materials Used in This Study: 3CL Protease (Millipore Sigma, Mpro, 3CL Protease from coronavirus SARS-COV-2, SAE0172), Cathepsin B Protease (Millipore Sigma, Cathepsin B, Human Liver, 219362-50UG), Caspase Protease (Enzo, Caspase-3 human, recombinant active, ALX-201-059-U025), MMP-7 Protease (Enzo. MMP-7 catalytic domain, human, recombinant, BML-SE181-0010). Bovine serum albumin (catalog no. A9418-5G).TABLE 5. Peptide sequences used in this studyTABLE 6. Triblock conjugate used in this study.EXAMPLE ASPECTS

[0287] Example 1 : A system for regulating enzyme function, the system comprising: a) an enzyme; b) an inhibitor of said enzyme; and c) a target recognition sequence linked to the inhibitor, wherein, when the target recognition sequence interacts with a target analyte, the inhibitor releases the enzyme and the enzyme is activated.

[0288] Example 2: The system of any examples herein, particularly Example 1, wherein the activated enzyme produces a detectable signal and / or performs a therapeutic function.

[0289] Example 3: The system of any examples herein, particularly Example 2, wherein the enzyme is thrombin, carbonic anhydrase, luciferase, beta-galactosidase, beta-lactamase, lactoperoxidase, horseradish peroxidase, granzyme B, trypsin, L-asparaginase. pegademase bovine, alglucerase, or streptokinase.

[0290] Example 4: The system of any examples herein, particularly Examples 1-3, wherein the inhibitor comprises one or more aptamers, one or more small molecules, one or more peptides, one or more antibodies, or any combination thereof.

[0291] Example 5: The system of any examples herein, particularly Example 4, wherein the enzyme is thrombin, and wherein the inhibitor comprises thrombin binding aptamer 15.

[0292] Example 6: The system of any examples herein, particularly Examples 1-5, further comprising an enzyme binding moiety linked to the target recognition sequence, and wherein the enzyme binding moiety comprises one or more aptamers, one or more small molecules, one or more peptides, one or more antibodies, or any combination thereof.

[0293] Example 7: The system of any examples herein, particularly Example 6, wherein the inhibitor and the enzyme binding moiety are linked to opposite ends of the target recognition sequence.

[0294] Example 8: The system of any examples herein, particularly Example 6, wherein the enzyme is thrombin, and wherein the enzyme binding moiety comprises thrombin binding aptamer 29.

[0295] Example 9: The system of any examples herein, particularly Examples 1-8, wherein the target recognition sequence comprises nucleic acid, an aptamer, a DNAzyme. a carbohydrate, or a peptide sequence.

[0296] Example 10: The system of any examples herein, particularly Examples 1-9, wherein interaction between the target recognition sequence and the target analyte induces a conformational change in the inhibitor, thereby causing the inhibitor to release the enzyme.

[0297] Example 11: The system of any examples herein, particularly Examples 1-10, wherein the target analyte binds to the target recognition sequence.

[0298] Example 12: The system of any examples herein, particularly Example 11, wherein the target recognition sequence comprises a first nucleic acid sequence, and wherein the target analyte comprises a second nucleic acid sequence that is at least partially complementary to the first nucleic acid sequence.

[0299] Example 13: The system of any examples herein, particularly Example 11, wherein the target recognition sequence comprises an aptamer, a DNAzyme, or a peptide, and wherein the target analyte is a small molecule, ion, or peptide.

[0300] Example 14: The system of any examples herein, particularly Examples 1-13, wherein the target analyte cleaves the target recognition sequence.

[0301] Example 15: The system of any examples herein, particularly Example 14, wherein the target recognition sequence comprises a peptide sequence, and wherein the target analyte is a protease.

[0302] Example 16: The system of any examples herein, particularly Examples 1-15, wherein the target analyte induces a conformational change in the target recognition sequence.

[0303] Example 17: The system of any examples herein, particularly Examples 1-16, wherein the target analyte indicates a disease or disorder.

[0304] Example 18: A method of regulating enzyme function, the method comprising providing the system of any examples herein, particularly Example 1 - 17, to a sample or subj ect.

[0305] Example 19: A method of detecting a target analyte in a sample, the method comprising: a) providing the system of any examples herein, particularly Examples 1-17, to the sample, wherein the activated enzyme produces a detectable signal; and b) detecting the detectable signal, thereby detecting the target analyte.

[0306] Example 20: The method of any examples herein, particularly Example 19, wherein the sample is a buffer sample, cell sample, a tissue sample, a biological fluid sample, or a cell lysate sample.

[0307] Example 21: The method of any examples herein, particularly Examples 19-20, further comprising observing and / or imaging the sample.

[0308] Example 22: The method of any examples herein, particularly Examples 19-21, wherein the enzyme is thrombin; wherein the method further comprises adding fibrinogen to the sample; and wherein the detectable signal is fibrinogen clots.

[0309] Example 23: The method of any examples herein, particularly Example 19-22, wherein presence of the detectable signal indicates presence of the target analyte, and wherein absence of the detectable signal indicates absence of the target analyte.

[0310] Example 24: A method of observing and / or monitoring a disease or disorder in a subject in need thereof, the method comprising administering to the subject the system of any examples herein, particularly Examples 1-17, wherein the target analyte indicates a disease or disorder and the activated enzy me produces a detectable signal.

[0311] Example 25: The method of any examples herein, particularly Example 24. further comprising observing and / or imaging the subject and / or a sample collected from the subject.

[0312] Example 26: The method of any examples herein, particularly Example 25, wherein the sample is a buffer sample, a cell sample, a tissue sample, a biological fluid sample, or a cell lysate sample.

[0313] Example 27: The method of any examples herein, particularly Examples 25-26. wherein the enzy me is thrombin; wherein the method further comprises adding fibrinogen to the sample; and wherein the detectable signal is fibrinogen clots.

[0314] Example 28: The method of any examples herein, particularly Example 24-27, wherein presence of the detectable signal indicates presence of the target analyte, and wherein absence of the detectable signal indicates absence of the target analyte.

[0315] Example 29: The method of any examples herein, particularly Examples 24-28, wherein, upon detection of the detectable signal, one or more treatments for the disease or disorder are administered to the subject.

[0316] Example 30: The method of any examples herein, particularly Examples 24-29, wherein the enzyme is activated only in a region affected by the disease or disorder.

[0317] Example 31: The method of claim 30, wherein the disease or disorder is cancer or a solid tumor, and wherein the enz me is activated only in or near cancerous cells or tumor cells.

[0318] Example 32: The method of any examples herein, particularly Example 30, wherein the disease or disorder is a bacterial, viral, fungal, or parasitic infection, and wherein the enzyme is activated only in or near infected cells.

[0319] Example 33: The method of any examples herein, particularly Examples 24-32, wherein the activated enzyme also performs a therapeutic function.

[0320] Example 34: A method of treating and / or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject the system of any examples herein, particularly Examples 1-17, wherein the target analyte indicates a disease or disorder and the activated enzyme performs a therapeutic function.

[0321] Example 35: The method of any examples herein, particularly Example 34, wherein the enzyme is activated only in a region affected by the disease or disorder.

[0322] Example 36: The method of any examples herein, particularly Example 35. wherein the disease or disorder is cancer or a solid tumor, and wherein the enzyme is activated only in or near cancerous cells or tumor cells.

[0323] Example 37: The method of any examples herein, particularly Example 35, wherein the disease or disorder is a bacterial, viral, fungal, or parasitic infection, and wherein the enzyme is activated only in or near infected cells.

[0324] Example 38: The method of any examples herein, particularly Example 34-37, wherein the enzy me is granzyme B or try psin.

[0325] Example 39: The method of any examples herein, particularly Examples 34-38, wherein the activated enzyme also produces a detectable signal.

[0326] Example 40: The method of any examples herein, particularly Example 39, further comprising observing and / or imaging the subject and / or a sample collected from the subject.

[0327] Example 41: The method of any examples herein, particularly Example 40, wherein the sample is a buffer sample, a cell sample, a tissue sample, a biological fluid sample, or a cell lysate sample.

[0328] Example 42: The method of any examples herein, particularly Example 39-41, wherein presence of the detectable signal indicates presence of the target analyte, and wherein absence of the detectable signal indicates absence of the target analyte.

[0329] Example 43: The method of any examples herein, particularly Examples 39-42, wherein, upon detection of the detectable signal, one or more additional treatments for the disease or disorder are administered to the subject.

[0330] Any patents, applications and publications as listed throughout this document are hereby incorporated by reference in their entirety herein.

Claims

What is claimed is:

1. A system for regulating enzyme function, the system comprising: a) an enzyme; b) an inhibitor of said enzy me; and c) a target recognition sequence linked to the inhibitor, wherein, when the target recognition sequence interacts with a target analyte, the inhibitor releases the enzyme and the enzyme is activated.

2. The system of claim 1, wherein the activated enzyme produces a detectable signal and / or performs a therapeutic function.

3. The system of claim 2, wherein the enzyme is thrombin, carbonic anhydrase, luciferase, beta-galactosidase, beta-lactamase, lactoperoxidase, horseradish peroxidase, granzyme B, trypsin. L-asparaginase, pegademase bovine, alglucerase, or streptokinase.

4. The system of claim 1, wherein the inhibitor comprises one or more aptamers, one or more small molecules, one or more peptides, one or more antibodies, or any combination thereof.

5. The system of claim 4, wherein the enzyme is thrombin, and wherein the inhibitor comprises thrombin binding aptamer 15 (SEQ ID NO: 1).

6. The system of claim 1, further comprising an enzyme binding moiety linked to the target recognition sequence, and wherein the enzyme binding moiety comprises one or more aptamers, one or more small molecules, one or more peptides, one or more antibodies, or any combination thereof.

7. The system of claim 6. wherein the inhibitor and the enzyme binding moiety are linked to opposite ends of the target recognition sequence.

8. The system of claim 6, wherein the enzyme is thrombin, and wherein the enzy me binding moiety comprises thrombin binding aptamer 29 (SEQ ID NO: 2).

9. The system of claim 1, wherein the target recognition sequence comprises nucleic acid, an aptamer, a DN Az me. a carbohydrate, or a peptide sequence.

10. The system of claim 1, wherein interaction between the target recognition sequence and the target analyte induces a conformational change in the inhibitor, thereby causing the inhibitor to release the enzyme.

11. The system of claim 1, wherein the target analyte binds to the target recognition sequence.

12. The system of claim 11, wherein the target recognition sequence comprises a first nucleic acid sequence, and wherein the target analyte comprises a second nucleic acid sequence that is at least partially complementary to the first nucleic acid sequence.

13. The system of claim 11, wherein the target recognition sequence comprises an aptamer, a DNAzyme. or a peptide, and wherein the target analyte is a small molecule, ion, or peptide.

14. The system of claim 1, wherein the target analyte cleaves the target recognition sequence.

15. The system of claim 14, wherein the target recognition sequence comprises a peptide sequence, and wherein the target analyte is a protease.

16. The system of claim 1, wherein the target analyte induces a conformational change in the target recognition sequence.

17. The system of claim 1, wherein the target analyte indicates a disease or disorder.

18. A method of detecting a target analyte in a sample, the method comprising: a) providing the system of claim 1 to the sample, wherein the activated enzyme produces a detectable signal: and b) detecting the detectable signal, thereby detecting the target analyte.

19. The method of claim 18, wherein the sample is a buffer sample, a cell sample, a tissue sample, a biological fluid sample, or a cell lysate sample.

20. A method of treating and / or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject the system of claim 1, wherein the target analyte indicates a disease or disorder and the activated enzyme performs a therapeutic function.

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