Methods and compositions related to nucleic acid-based fluorescent nanocluster probes

Abstract

Probes of the invention can indicate the presence of an analyte, the probe comprising a nucleic acid sequence and a fluorescent metal nanocluster associated with the nucleic acid sequence. These probes can be modified in a multitude of ways which can allow them to be used in a variety of detection assays. The probes can also be combined in the form of a composition with various components, which allow the probes to be used to detect an analyte (target). This target can be a nucleic acid, a protein, or a small molecule, for example.

Description

[0001] Attorney Docket No. 10046-616W01

[0002] METHODS AND COMPOSITIONS RELATED TO NUCLEIC ACID-BASED FLUORESCENT NANOCLUSTER PROBES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 696,165, filed September 18, 2024, which is incorporated by reference herein in its entirety.

[0005] GOVERNMENT SUPPORT CLAUSE

[0006] This invention was made with government support under Grant no. CBET2029266 awarded by the National Science Foundation. The government has certain rights in the invention.

[0007] REFERENCE TO SEQUENCE LISTING

[0008] The sequence listing submitted on September 18, 2025, as an .XML file entitled “10046-616W01_ST26.xml” created on September 17, 2025, and having a file size of 40,790 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0009] BACKGROUND

[0010] Although Fluorescent Resonance Energy Transfer (FRET) probes have enabled many sensitive and highly specific assays, FRET probes have a number of limitations, particularly in the field of detection of nucleic acids. They are expensive to design and manufacture, and they are difficult to multiplex. They still cost ~$300 for 1 nanomole of ssRNA-based or 6.5 nanomole ssDNA-based FRET reporters. Besides, most FRET probes only allow for singlechannel emission measurement (single color) due to their intrinsic donor-quencher system, which also causes relatively high background signal. There exists in the art a need for improved probes for detecting nucleic acid cleavage events rapidly, sensitively, reliably and quantitatively. Ideal probes would give rise to minimal background signal and be easily and inexpensively prepared.

[0011] Fluorescent metal nanoclusters, such as DNA-based silver nanoclusters (DNA / AgNCs), are emerging fluorescence tools in imaging, catalysis, sensing, and biomedicine. DNA / AgNCs display superior optical performance since their size is close to the Fermi wavelength. DNA / AgNCs possess unique features, including high fluorescence quantum yields and stability, biocompatibility, facile synthesis, and low toxicity, which are requisite for fluorescent probes. The fluorescent emission of DNA / AgNCs can cover the violet Attorney Docket No. 10046-616W01

[0012] (UV, 300 nm) to near-infrared (NIR, 950 nm) region by varying the DNA sequences, lengths, and structures or by modifying the environmental factors (such as buffer, pH, metal ions, macromolecular polymers, and small molecules) (Yang M, Chen X, Su Y, Liu H, Zhang H, Li X and Xu W (2020) The Fluorescent Palette of DNA-Templated Silver Nanoclusters for Biological Applications. Front. Chem. 8:601621).

[0013] What is needed in the art are non-FRET-based reporter molecules. This need and others are at least partially satisfied by the present disclosure.

[0014] SUMMARY

[0015] In some aspects, disclosed herein is a probe including: a nucleic acid sequence, wherein said nucleic acid sequence includes a cleavage site including at least one RNA nucleotide and / or a non-nucleic acid molecule; and a fluorescent metal nanocluster associated with the nucleic acid sequence; wherein the fluorescent metal nanocluster produces a detectable signal upon cleavage of the nucleic acid sequence by a catalytic agent.

[0016] In some aspects, also disclosed herein is a composition including any of the disclosed probes and a buffer that stabilizes the fluorescent metal nanocluster and associated nucleic acid sequence.

[0017] In some aspects, also disclosed herein is a method of detecting activity of a catalytic agent in a sample, the method including: a) providing any of the disclosed probes to the sample; and b) detecting the detectable signal, thereby detecting activity of the catalytic agent.

[0018] 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.

[0019] BRIEF DESCRIPTION OF DRAWINGS

[0020] FIGURES 1A-1C depict an example modified Subak probe. FIG. 1A shows a modified Subak probe for detection of a small molecule (e.g., a therapeutic drug called clozapine). Combining an aptamer that targets a small molecule and a DNAzyme that uses a Pb2+or Mg2+as cofactors, an aptazyme is created, which acquires RNA cleaving capability when small-molecule target and metal cofactor are present in the sample and bind aptazyme together. The active aptazyme then cuts RNA-containing Subak and turn the sample from green to red. FIG. IB shows, in a control experiment, modified Subak (an RNA-containing Subak) was tested for RNase activity detection, where an rU is incorporated in the loop of Subak. A red emission Attorney Docket No. 10046-616W01 peak is seen upon cleaving this rU-containing Subak using an RNase A / Tl mix solution. FIG. 1C shows, in another control experiment, when 10-23 DNAzyme is activated by Mg2+ions, it can cleave Subak probe and show a red color. Representative sequences can be found in SEQ ID NO: 2 and 12-13.

[0021] FIGURE 2 depicts Subak probes based on highly fluorescent silver nanoclusters are cost-efficient, non-FRET, color-changing probes for nuclease activity detection.

[0022] FIGURES 3 depicts an aptazyme with a non-FRET probe.

[0023] FIGURE 4 depicts Subak (SEQ ID NO: 1) with ribonucleotide mutation showed brighter red color by cleavage upon RNase. Representative sequences can be found in SEQ ID NOS: 3-9.

[0024] FIGURE 5 depicts Subak with ribonucleotide modification can be cleaved by 10-23 DNAzyme. Chimeric Subak-1 is SEQ ID NO: 10 (only the DNA arm binding portion shown), the DNA arm is SEQ ID NO: 11, and green chimeric Subak-1 is SEQ ID NO: 10.

[0025] FIGURE 6 depicts Mg-specific 10-23 DNAzyme cleaved Subak and showed color change from green to red. The stem-cut Subak is SEQ ID NO: 10, and the loop-cut Subak is SEQ ID NO: 12.

[0026] FIGURE 7 depicts optimizing of Subak probe with various DNAzymes for biosensing applications (see Claire E. McGhee, et al., ACS Central Science 2021 7 (11), 1809-1820). A representative sequence can be found in SEQ ID NO: 14.

[0027] FIGURES 8A-8B depict that a photocleavable (PC) linker can be incorporated in Subak for UV-induced cleavage (SEQ ID NO: 1 shown, SEQ ID NO: 15 is representative of a sequence with a linker).

[0028] FIGURE 9 depicts a schematic overview of Subak constructs and their detection modalities. Subak reporters can be adapted for RNase (rSubak-a), CRISPR-Casl3a (rSubak- P), or photo-cleavage (pcSubak) detection. rSubak-a enables ratiometric fluorescence readout and supports visual detection via absorbance change. The system is also compatible with biological matrices such as human serum, highlighting its potential for field-deployable sensing.

[0029] FIGURES 10A-10C depict quantification of RNase A / Tl using rSubak. FIG. 10A shows concentration-dependent fluorescence spectra were measured under 280 nm excitation. FIG. 10B shows kinetic fluorescence emission spectra of rSubak in response to various amounts of RNase A / Tl. The red fluorescence (E25) was measured at 625 nm under 280 nm excitation. The lines represent the mean values, while the shadings represent the standard deviations (n = 3 replicates). FIG. IOC shows single-color (E25) and ratiometric sensing Attorney Docket No. 10046-616W01

[0030] (I625 / I530) results of purified rSubak on RNase A / Tl. The green fluorescence (I530) was measured at 540 nm under 280 nm excitation, while the red fluorescence (L525) was measured at 625 nm under 280 nm excitation.

[0031] FIGURES 11A-11B depict thermal stability of Subak probes and ratiometric performance after PCR cycling. FIG. 11 A shows relative green fluorescence remaining in Subak-1 and Subak-2 after exposure to PCR thermal cycling (95°C and 60°C). Fluorescence intensities were measured at defined cycle intervals, normalized to the initial (cycle 0) fluorescence. A 50% threshold is indicated for reference. FIG. 11B shows the ratiometric signal (I625 / I530) of Subak probes following thermal cycling and subsequent DNase I digestion. Data show improved ratiometric contrast as the number of PCR cycles increases.

[0032] FIGURE 12 depicts detection of viral ssRNA using the rSubak probewith CRISPR- Casl3a system. rSubak cleavage by Casl3a occurs in response to specific viral RNA targets, leading to color-switching of DNA-templated silver nanoclusters.

[0033] FIGURES 13A-13C depict a performance comparison of rSubak with conventional RNaseAlert probe. The probes were used for detection of SARS-CoV-2 (FIG. 13A), A / H5N1 (FIG. 13B), and Measles virus (MV, FIG. 13C) ssRNA using RNaseAlert. rSubak achieves markedly lower limits of detection (LoD) across all three viral targets, does not require postsynthesis purification, offers ratiometric readout, and reduces probe cost from ~$75.60 / nmol (RNaseAlert) to ~$l / nmol.

[0034] DETAIEED DESCRIPTION

[0035] General Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0037] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will 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 Attorney Docket No. 10046-616W01 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.

[0038] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0039] As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0040] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0041] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0042] By “reduce,” or “abrogate,” (used interchangeably) or other forms of the word, such as “reducing” or “reduction,” or “abrogating” or “abrogation” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0043] By “increase” or other forms of the word, such as “increasing,” is meant raising or elevating. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. Attorney Docket No. 10046-616W01

[0044] As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g, cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0045] “Control” refers to a sample or standard used for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy subject (or a plurality of healthy subjects), such as a subject or subjects not expected or known to have a particular polymorphism. In additional embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample or plurality of such samples), or group of samples that represent baseline or normal values. A positive control can be an established standard that is indicative of a specific methylated nucleotide. In some embodiments a control nucleic acid is one that lacks a particular methylated nucleotide, and is used in assays for comparison with a test nucleic acid, to determine if the test nucleic acid includes the methylated nucleotide.

[0046] “Detecting” is used herein to identify the existence, presence, or fact of something. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein. For example, included herein are methods of detecting a nucleic acid molecule in sample. Detection can include a physical readout, such as fluorescence output.

[0047] “Enhancer Sequence” refers to a nucleotide sequence that when placed in proximity to another nucleic acid molecule having templated metal nanoclusters increases the fluorescence intensity of the metal nanocluster when exposed to excitation light. Exemplary enhancer sequences are known in the art and disclosed herein.

[0048] “Excitation Light” refers to light of any wavelength that is capable of causing template metal nanoclusters to fluoresce. Non-limiting examples of excitation light include visible light, ultraviolet and near infrared light.

[0049] The term “hybridization” is defined as forming base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule, for example. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+concentration) of the hybridization buffer will determine Attorney Docket No. 10046-616W01 the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11).

[0050] An “isolated” biological component (such as a nucleic acid molecule) has been substantially separated, produced apart from, or purified away from other biological components. Nucleic acid molecules which have been “isolated” include nucleic acids molecules purified by standard purification methods, as well as those chemically synthesized. Isolated does not require absolute purity, and can include nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated.

[0051] A “nucleation sequence” is a sequence of nucleotides capable of binding or associating with metal atoms to form template metal nanoclusters. The portion of a nucleic acid molecule including a nucleation sequence of nucleotides is referred to as the “nucleation portion” of the nucleic acid molecule. Exemplary nucleation sequences are known and provided herein. Specific nucleation sequences that are useful for interacting with metal nanoclusters and forming DNA templated metal nanoclusters are disclosed herein. Examples of metal nanoclusters for use as fluorescent reporters, and methods of producing templated metal nanoclusters on DNA oligonucleotides are known. See, e.g., U.S. Patent Publication No. US20110212540, incorporated by reference herein in its entirety, and U.S. Publication No. US20140349289, incorporated herein by reference.

[0052] A “nucleic acid” is a deoxyribonucleotide or ribonucleotide polymer, which can include analogues of natural nucleotides that hybridize to nucleic acid molecules in a manner similar to naturally occurring nucleotides. In a particular example, a nucleic acid molecule is a single stranded (ss) DNA or RNA molecule, such as a probe or primer. In another particular example, a nucleic acid molecule is a double stranded (ds) nucleic acid, such as a target nucleic acid. Examples of modified nucleic acids are those with altered backbones, such as peptide nucleic acids (PNA).

[0053] The major nucleotides of DNA are deoxyadenosine 5 '-triphosphate (dATP or A), deoxyguanosine 5 '-triphosphate (dGTP or G), deoxy cytidine 5 '-triphosphate (dCTP or C) and deoxythymidine 5'-triphosphate (dTTP or T). The major nucleotides of RNA are adenosine 5'- triphosphate (ATP or A), guanosine 5 '-triphosphate (GTP or G), cytidine 5 '-triphosphate (CTP or C) and uridine 5 '-triphosphate (UTP or U).

[0054] Nucleotides include those nucleotides containing modified bases, modified sugar moieties and modified phosphate backbones, as known in the art. Attorney Docket No. 10046-616W01

[0055] Examples of modified base moieties which can be used to modify nucleotides at any position on its structure include, but are not limited to: 5 -fluorouracil, 5 -bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5 -carboxymethylaminomethyl -2 -thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6- isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5 -methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5 '-methoxy carboxymethyluracil, 5 -methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5 -oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5 -methyl - 2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5 -oxyacetic acid methylester, uracil-5 -oxyacetic acid, 5 -methyl -2 -thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine .

[0056] Examples of modified sugar moieties which may be used to modify nucleotides at any position on its structure include, but are not limited to: arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof.

[0057] The term “complementary binding” as used herein occurs when the base of one nucleic acid molecule forms a hydrogen bond to the base of another nucleic acid molecule. Normally, the base adenine (A) is complementary to thymidine (T) and uracil (U), while cytosine (C) is complementary to guanine (G). For example, the sequence 5'-ATCG-3' of one ssDNA molecule can bond to 3'-TAGC-5' of another ssDNA to form a dsDNA. In this example, the sequence 5'-ATCG-3' is the reverse complement of 3'-TAGC-5'. Nucleic acid molecules can be complementary to each other even without complete hydrogen-bonding of all bases of each molecule. For example, hybridization with a complementary nucleic acid sequence can occur under conditions of differing stringency in which a complement will bind at some but not all nucleotide positions.

[0058] A “polymorphism” is a variation in a gene sequence. The polymorphisms can be those variations (DNA sequence differences, e.g., substitutions, deletions, or insertions) which are generally found between individuals or different ethnic groups and geographic locations which, while having a different sequence, produce functionally equivalent gene products. Typically, the term can also refer to variants in the sequence which can lead to gene products that are not functionally equivalent. Polymorphisms also encompass variations which can be classified as Attorney Docket No. 10046-616W01 alleles and / or mutations which can produce gene products which may have an altered function. Polymorphisms also encompass variations which can be classified as alleles and / or mutations which either produce no gene product or an inactive gene product or an active gene product produced at an abnormal rate or in an inappropriate tissue or in response to an inappropriate stimulus. Alleles are the alternate forms that occur at the polymorphism.

[0059] Polymorphisms can be referred to, for instance, by the nucleotide position at which the variation exists, by the change in amino acid sequence caused by the nucleotide variation, or by a change in some other characteristic of the nucleic acid molecule or protein that is linked to the variation.

[0060] “Probes,” as used herein, refer to short nucleic acid molecules, usually DNA or RNA oligonucleotides, typically of about 3-150 nucleotides in length, and more specifically, about 6-100 nucleotides in length, used to detect the presence of a complementary target nucleic acid or small molecule in a sample. All or a portion of a probe can be annealed to a complementary target nucleic acid strand by nucleic acid hybridization to form a hybrid between the probe and the target DNA strand. The probes are associated with metal nanoclusters. Therefore, nucleic acid-metal nanocluster probes can be used to identify a target nucleic acid molecule, wherein the sequence of the probe is specific for the target nucleic acid molecule, for example so that the probe will hybridize to the target nucleic acid molecule under very high stringency hybridization conditions.

[0061] Typically, the nucleic acid portion of the probe includes at least about 6 contiguous nucleotides, such as at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50 contiguous nucleotides, that are complementary to a target nucleic acid molecule, such as 20-70 nucleotides, 20-60 nucleotides, 20-50 nucleotides, 20-40 nucleotides, or 20-30 nucleotides. Probes can also be of a maximum length, for example no more than 20, 25, 25, 40, 50, 75 or 100 nucleotides in length. The specificity of a particular probe typically increases with an increase in the number of complementary nucleotides on the probe.

[0062] The probe can also include additional nucleotides that are not complementary to the target nucleic acid molecule. The additional nucleotides can be used, for example, for detection of the probe in a sample. In several embodiments, the probes disclosed herein include a hybridization portion that is complementary to a test nucleic acid sequence, and a cleavable portion (that can associate with metal nanoclusters) or an enhancer portion (that can enhance the fluorescence of metal nanoclusters associated with the cleavable portion). The additional nucleotides can be located 5' or 3' of the hybridization nucleotides. Attorney Docket No. 10046-616W01

[0063] Methods for preparing and using nucleic acid probes are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990).

[0064] The identity / similarity between two or more nucleic acid sequences is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.

[0065] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0066] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI web site. BLASTN is used to compare nucleic acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

[0067] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166=1554* 100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are Attorney Docket No. 10046-616W01 rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20- nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15=20* 100=75). One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above.

[0068] A “sample,” such as a biological sample, is a sample obtained from a subject. As used herein, biological samples include all clinical samples useful for detection of a methylated nucleotide, including, but not limited to, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; urine; sputum; or CVS samples. In a particular example, a sample includes blood obtained from a human subject, such as whole blood or serum.

[0069] A “test nucleic acid molecule” refers to a nucleic acid molecule whose detection, quantitation, qualitative detection, characterization, or a combination thereof, is intended. For example, the test nucleic acid molecule can be a defined region or particular portion of a nucleic acid molecule, for example a portion of a genome (such as a gene or a region of DNA or RNA containing a gene or portion thereof of interest). The nucleic acid molecule need not be in a purified form. Various other nucleic acid molecules can also be present with the test nucleic acid molecule. For example, the test nucleic acid molecule can be a specific nucleic acid molecule (which can include RNA or DNA), for which the detection of a particular polymorphism is intended. In some examples, a test nucleic acid includes a viral nucleic acid molecule, or a bacterial nucleic acid molecule. Purification or isolation of the test nucleic acid molecule, if needed, can be conducted by methods known to those in the art, such as by using a commercially available purification kit or the like.

[0070] By “contacting” is meant placement in direct physical association, for example solid, liquid or gaseous forms. Contacting includes, for example, direct physical association of fiilly- and partially-solvated molecules.

[0071] A “metal nanocluster” is a collection of small numbers (e.g., 2-30 atoms) of noble metal atoms (e.g., gold or silver atoms) with physical sizes close to the Fermi wavelength of an electron (~3 nm for gold and silver). The metal ions can have affinity for nitrogen atoms on DNA, including the N3 of cytosine and the N7 of guanine. Metal nanoclusters for use with the disclosed embodiments are fluorescent, that is, they have the ability to emit light of a particular wavelength (emission wavelength) when exposed to light of another wavelength (excitation wavelength). Attorney Docket No. 10046-616W01

[0072] Specific nucleotide sequence (“nucleation sequences”) that are useful for interacting with metal nanoclusters and forming DNA templated metal nanoclusters are disclosed herein. Examples of metal nanoclusters for use as fluorescent reporters, and methods of producing templated metal nanoclusters on DNA oligonucleotides are known. See, e.g., U.S. Pat. App. Pub. 2011 / 0212540 entitled “Probe and Method for DNA Detection”, which was filed Feb. 22, 2011, and is incorporated by reference herein in its entirety. See also Richie et al., “Ag Nanocluster Formation using a cytosine oligonucleotide template,” J Phys Chem C, 111, 175- 181, 2006, which is incorporated by reference herein in its entirety.

[0073] DNA-based enzymes, also known as deoxyribozymes or “DNAzymes”, are singlestranded DNA molecules with catalytic activity. DNAzymes are characterized by two domains: catalytic and substrate binding domains. However, their sequences can be varying. There are two main types: “10-23” and “8-17” DNAzymes.

[0074] “Ribonuclease” (commonly abbreviated “RNase”) is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and include several sub-classes within the EC 2.7 (for the phosphorolytic enzymes) and 3.1 (for the hydrolytic enzymes) classes of enzymes.

[0075] General Description of the Invention

[0076] Disclosed herein are probes which can indicate the presence of an analyte, the activity of an enzyme, and / or a change in environmental conditions, the probe including a nucleic acid sequence and a fluorescent metal nanocluster associated with the nucleic acid sequence. An example of this can be seen in FIGS. 1A-1C. These probes can be modified in a multitude of ways which can allow them to be used in a variety of detection assays. Examples of some of these ways can be seen in TABLE 1. The probes can also be combined in the form of a composition with various components which were not previously known in the prior art, which allow the probes to be used to detect an analyte. This analyte can be a nucleic acid, a protein, or a small molecule, for example. Below follows a general description of these probes. Modifications and additions to the composition as discussed after that. Attorney Docket No. 10046-616W01

[0077] TABLE 1. Various Uses for Subak Probes Attorney Docket No. 10046-616W01

[0078] Fluorescent Metal Nanocluster Probes

[0079] The probes disclosed herein include two parts: a fluorescent metal nanocluster and an associated nucleic acid sequence. These nucleic acid-metal nanoclusters are multicolor fluorophores associated with a nucleic acid whose emission fluorescence can be tuned by rearranging the nucleobases surrounding the clusters. The encapsulated metal nanocluster lights up significantly with a shift in the emission peaks. This shift in emission peak can be accomplished by changing the structure of the probe, such as by cleavage. Examples of these probes can be found, for example, in Hong et al., A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion. Nat Nanotechnol. 2024 Jun;19(6):810-817, herein incorporated by reference in its entirety for its disclosure concerning “Subak” probes.

[0080] The nucleic acid-metal nanocluster probes disclosed herein are highly fluorescent (with quantum yields up to 0.9 and extinction coefficients ~105Macin'1) (Petty 2018; Obliosca 2013; Neascu 2020), multicolor (emission peaks ranging from 300 - 950 nm) (Obliosca 2013; Choi 2012), activatable (Yeh 2010; Obliosca 2014; Petty 2013), color tunable (Petty 2014; Del Bonis-O’Donnell 2019; Cerretani 2019), environmentally sensitive (Obliosca 2013; Choi 2012), and biocompatible Sharma 2012). DNA or RNA can template the growth of few-atom silver nanoclusters (Ags, Agio or Agio) (Huard 2018; Petty 2016; Cerretani 2019), creating a palette of organic-inorganic composite nanomaterial fluorophores (FIG. IB). Nucleic acidmetal nanocluster probes such as DNA / AgNCs are unique probes, as the photophysical properties of metal nanoclusters, such as silver clusters can reflect subtle changes in their surrounding ligand (i.e., nucleobase) environment, making them superior sensors for a number of applications (Obliosca 2013), including DNA (Obliosca 2014; Sharma 2010), SNP (Yeh 2012), DNA methylation (Chen 2015), and enzyme activity (Juul 2015) detection. The nucleic acid-metal nanocluster probes disclosed herein are alternatively referred to as “Digest-on” probes or “Subak probes.”

[0081] The disclosed embodiments take advantage of the differential fluorescent properties of metal (e.g., silver) nanoclusters when the nanoclusters are brought near different nucleic acid sequences. The metal of the templated metal nanoclusters can be a noble metal, such as silver, gold, or copper. Particularly envisioned herein is a silver nanocluster (AgNC). Nucleic acid- templated silver nanoclusters can emit colored light. The color of the emitted light was found to depend on the particular nucleic acid sequence it is associated with. Importantly, this emitted light can shift when different nucleic acids are brought in contact with the AgNC. Silver nanoclusters are groups of from about 2 to about 30 silver atoms that are about less than 3 nm in size with the properties of good fluorescence, good photostability, and electroluminescence. Attorney Docket No. 10046-616W01

[0082] These silver nanoclusters can function as fluorescence reporters of nucleic acid digestion or differential hybridization.

[0083] To form metal nanoclusters on DNA, positively charged metal ions (e.g., Ag+atoms) are first attached to ssDNA spontaneously in solution. Then, a reductant (e.g., sodium borohydride) is added to reduce the charge of the atoms (e.g., Ag+to Ag(0)), after which metal atom “clusters” will form. The ssDNA prevents the metal cluster “from growing out of control”. Clusters that become a “nanoparticle” (size >5 nm) are not fluorescent.

[0084] Examples of metal nanoclusters for use as fluorescent reporters, and methods of producing templated metal nanoclusters on DNA oligonucleotides are known. See, e.g., U.S. Pat. App. Pub. 2011 / 0212540 entitled “Probe and Method for DNA Detection”, which was filed Feb. 22, 2011, and is incorporated by reference herein in its entirety. See also Richie et al., “Ag Nanocluster Formation using a cytosine oligonucleotide template,” J Phys Chem C, 111, 175-181, 2006, which is incorporated by reference herein in its entirety. The basis for the operation of the templated metal nanoclusters is a controlled conversion of DNA-templated silver nanoclusters between a dark, non-emissive state, which is their state when not associated with an enhancer sequence, and a bright, emissive state when associated with the enhancer sequence. Unlike prior use of metal nanoclusters, the present method involves tuning the fluorescent emission properties of the metal nanoclusters (e.g., a wavelength shift of 60-70 nm) by altering the relative positions of the nanoclusters to the and the enhancer sequence.

[0085] The disclosure of the following references and their description of templated metal nanoclusters and their use and detection are incorporated by reference herein in its entirety: Petty et al., J American Chemical Society 2004, 126, 5207; Vosch et al., Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12616; Gwinn et al., Adv. Mater. 2008, 20, 279; Petty et al., Anal. Chem. 2011, 83, 5957; Sharma et al., Chem. Commun. 2010, 46, 3280; Sharma et al., Chem. Commun. 2011, 47, 2294; Neidig et al., J. Am. Chem. Soc. 2011, 133, 11837; and Yeh et al., Nano Eett. 2010, 10, 3106.

[0086] Noble metal nanoclusters, such as those made of silver, gold, copper, or other noble metals typically include collections of a number of metal atoms (approximately 2-30 atoms or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 atoms) with physical sizes close to the Fermi wavelength of an electron (e.g., about 0.5 nm for gold and silver). They behave like molecular systems and yield fluorescence emission in the UV-visible and infrared range. In some examples, oligonucleotide-templated silver nanoclusters (“DNA / AgNCs”), which are a versatile set of fluorophores that have been used for a variety of applications including live cell imaging, detection of specific metal ions, and Attorney Docket No. 10046-616W01 single-nucleotide variation identification. DNA / AgNCs can be biocompatible and can have better photostability than commonly used organic dyes. Unlike organic dyes and photoluminescent nanocrystals, they are subject to silver oxidation / reduction or nanocluster (“NC”) regrouping, which results in conversion among different NC species. These different species may provide different color emissions.

[0087] Also contemplated herein are composite nanoclusters made of more than one material, such as silver and copper.

[0088] Detection of fluorescence emission can be performed according to known methods, for example as described herein. The excitation light can be selected from the group consisting of ultraviolet light, visible light, near infrared light or a combination thereof. In a related aspect, the wavelength of excitation light is from 200 nm to 2000 nm (or 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 or 2000 nm).

[0089] The metal nanoclusters are associated with a nucleic acid to form the probe. The nucleic acid portion of the probe can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, 21,

[0090] 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 ,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

[0091] 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

[0092] 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,

[0093] 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 nucleotides long, or longer. In a particular embodiment, the nucleic acid is between 3-150 nucleotides long, or between 6-100 nucleotides long.

[0094] The nucleic acid portion of the probe can include RNA, DNA (including aforementioned nucleotide analogs), or some combination thereof. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5 methylcytosine (5 me C), 5 hydroxymethyl cytosine, xanthine, hypoxanthine, and 2 aminoadenine as well as modifications at the sugar or phosphate moieties. There are many varieties of these types of molecules available in the art and available herein. Modifications to the probe are discussed in more detail below.

[0095] The nucleic acid sequence further includes a cleavage site including at least one DNA nucleotide, at least one RNA nucleotide, and / or a non-nucleic acid molecule. For example, in Attorney Docket No. 10046-616W01 some aspects, the nucleic acid sequence can include DNA and the cleavage site can include at least one RNA nucleotide. In other aspects, the nucleic acid sequence can include RNA and the cleavage site can include at least one DNA nucleotide. In yet other aspects, the nucleic acid can include DNA and / or RNA and the cleavage site can include a non-nucleic acid molecule. In yet still other aspects, the nucleic acid can include DNA and the cleavage site can include a non-nucleic acid molecule and at least one RNA nucleotide. In yet still other aspects, the nucleic acid can include RNA and the cleavage site can include a non-nucleic acid molecule and at least one DNA nucleotide. The cleavage site can be cleaved by a catalytic agent. Both the cleavage site and the catalytic agent are discussed in further detail below.

[0096] Upon cleavage of the cleavage site, the fluorescent metal nanocluster can produce a detectable signal (e.g., a change in color). Notably, the use of a cleavage site which differs compositionally from the remainder of the nucleic acid sequence can improve the sensitivity and applicability of the probes. For example, incorporating RNA nucleotides the probes enables the detection of RNases and RNA viruses. This modification broadens the potential applications of the probes beyond DNases and DNA virus detection to include a wide range of targets, such as RNases and RNA viruses. Using a cleavage site which compositionally differs from the remainder of the probe can allow higher accuracy of the location of the cleavage and minimize off-target cleavage of the probe. This also allows detection of stimuli that do not cleave nucleotides, for example, proteases or environmental conditions like exposure to light. In some instances, this can also allow more specialized and / or unstable molecules to be incorporated into the cleavage site, as the remainder of the probe can be designed to provide a stabilizing effect to the cleavage site.

[0097] When incorporating RNA into the Subak design, we found that excessive RNA destabilizes the structure and weakens green fluorescence, so we limited RNA to the minimal regions required. Red emission increases when cleavage occurs precisely at the designed site, not through indiscriminate cutting. For instance, rSubak cleaved by RNase produces a stronger red signal than the DNA-only Subak cleaved by DNase, which can also destroy red-forming fragments. Although we have not tested a fully RNA probe, we expect it would behave differently due to distinct RNA / AgNC interactions. The key advantage of introducing RNA selectively at cleavage sites is precise control over the reaction, enabling more efficient red conversion, while rSubak still retains red conversion activity with DNase as well.

[0098] The nucleic acid portion of the probe can include secondary structure. An example of a hairpin can be seen in FIG. 3. It is noted that any probe which is capable of working with the metal nanoclusters can be used for the methods described herein, regardless of the shape. Attorney Docket No. 10046-616W01

[0099] Examples of secondary structure include, but are not limited to, double helices, stem-loop structures, hairpins, psuedoknots, and G-quadruplexes. It has been found that hairpin structures are particularly useful, and therefore the probes described herein can be used in a hairpin form, as shown in FIG. 5 by way of example. One specific example of a probe which can be used is a “Subak probe.” Subak probes can include SEQ ID NO: 1, for example, or a nucleic acid with one, two, three, four, five, six, seven, eight, nine, or ten or more changes in sequence as compared to SEQ ID NO: 1. For example, disclosed herein is a sequence with 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any one of SEQ ID NOS: 1-18. Examples include SEQ ID NOS: 2-18. Specifically contemplated are sequences which include RNA (SEQ ID NOS: 2-11, 13, and 16-18), and those with a photocleavable linker (SEQ ID NO: 15). SEQ ID NO: 2 represents a probe which can be used with an aptazyme as the catalytic agent. SEQ ID NOS: 3-9 and 16-18 represent probes which can be used with RNase as the catalytic agent. SEQ ID NOS: 10-14 represent probes which can be used with DNAzyme as the catalytic agent. SEQ ID NO: 15 represents a probe with a photocleavable linker which can be used with exposure to UV light as the catalytic agent. SEQ ID NO: 18 represents a probe which can be used with Casl3a as the catalytic agent. These modifications, as well as others, are discussed in more detail below.

[0100] As mentioned above, these probes can be modified in a variety of novel ways, and combined in the form of a composition with a variety of components which allow them to be used in a novel manner. Discussed below are modifications to nucleic acid-metal nanocluster probes, as well as compositions including nucleic acid-metal nanocluster probes.

[0101] Cleavage Sites and Catalytic Agents

[0102] In some aspects, the cleavage site can include at least one RNA nucleotide or at least one DNA nucleotide. For example, in some aspects, the cleavage site can include 1 or more RNA nucleotides or DNA nucleotides (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more). In some aspects, the cleavage site can include 6 or less RNA nucleotides or DNA nucleotides (e.g., 5 or less, 4 or less, 3 or less, 2 or less, 1 or less).

[0103] The cleavage site can include any amount of RNA nucleotides or DNA nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the cleavage site can include from 1 to 6 RNA nucleotides or DNA nucleotides (e.g., from 2 to 5, from 3 to 4, from 1 to 4, from 2 to 3, from 3 to 6, from 4 to 5). Attorney Docket No. 10046-616W01

[0104] An example of RNA bases in the probe can be seen in FIG. 5. When the cleavage site includes at least one RNA nucleotide or at least one DNA nucleotide, in some aspects, the catalytic agent can be a nuclease, a ribozyme, or a DNAzyme.

[0105] When a DNAzyme is used, it can cleave RNA or DNA (which are respectively denoted as an RNA-cutting DNAzyme and a DNA-cutting DNAzyme). In some aspects, the DNAzyme can be an aptazyme which is sensitive to an analyte. In other words, the DNAzyme can function as a biosensor, reacting in the presence of the analyte and then causing a detectable change (i.e., the detectable signal). Examples of DNAzymes which can be used with the present invention can be found in McConnell et al. (Biosensing with DNAzymes. Chem Soc Rev. 2021 Aug 21;50(16):8954-8994), herein incorporated by reference in its entirety for its teaching concerning DNAzymes. Ribonucleotide modifications can be strategically introduced in the loop region to facilitate enhanced accessibility for DNAzymes, as well as in the stem region, which includes the preferred cleavage sites for Subak.

[0106] The analyte to be detected using the probes disclosed herein can include any number of molecules which can interact with the aptazyme to bring about activation of the aptazyme. The analyte can include, for example, a nucleic acid, a small molecule, a protein, or a metal ion. Examples of metal ions include, but are not limited to, Pb2+, Cu+, Cu2+, UCh2+, Zn2+, Mg2+, Hg2+, Ag+, Ca2+, Tl3+, Cd2+, C+, Co2+, Ni2+, Na+, Li+, Fe2+, Fe3+, and Mn2+. Examples of molecules which can be detected include, but are not limited to, food contaminants, pathogens (viruses, bacteria, etc.), and various clinically relevant biomarkers. Examples of small molecules which can be detected include, but are not limited to, dopamine, adenosine, AMP, and ATP.

[0107] When the analyte is a nucleic acid, the target nucleic acid may not hybridize to the probe under hybridization conditions. Or, the probe may hybridize partially, but not fully. By “partially” is meant that the target nucleic acid may hybridize over a portion of the probe, or it may weakly hybridize (associate) with the probe. This partial or non-substantial hybridization may be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,

[0108] 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,

[0109] 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,

[0110] 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,

[0111] 79%, or 80% hybridization. “Hybridization conditions” is considered the standard conditions in which one nucleic acid molecule would hybridize to another. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Attorney Docket No. 10046-616W01

[0112] Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11).

[0113] When a nuclease is used, it can cleave RNA or DNA. In some aspects, the nuclease can be RNase or DNase. Monitoring RNase and DNase activity is critical in laboratory and manufacturing settings to ensure nuclease-free conditions, as uncontrolled nuclease activity can degrade nucleic acids and compromise experiments. These probes can be used for quality control of reagents, lab equipment, and solutions, ensuring reliability in RNA- and DNA-based experiments. These probes can enable routine contamination monitoring while maintaining high performance, making them valuable beyond mere activity measurement.

[0114] In some aspects, the nuclease can be a Cas protein, and the probe can be used in a CRISPR / Cas system for nucleic acid detection. In general, a CRISPR-Cas or CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).

[0115] In certain embodiments, a protospacer adjacent motif (PAM) or PAM-like motif directs binding of the effector protein complex as disclosed herein to the target locus of interest. In some embodiments, the PAM may be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM may be a 3' PAM (i.e., located downstream of the 5' end of the protospacer). The term “PAM” may be used interchangeably with the term “PFS” or “protospacer flanking site” or “protospacer flanking sequence”.

[0116] The CRISPR effector protein may recognize a 3' PAM. In certain embodiments, the CRISPR effector protein may recognize a 3' PAM which is 5'H, wherein H is A, C or U. In certain embodiments, the effector protein may be Leptotrichia shahii C2c2p. more preferably Leptotrichia shahii DSM 19757 C2c2, and the 3' PAM is a 5' H.

[0117] In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization Attorney Docket No. 10046-616W01 between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence may include RNA polynucleotides. The term “target RNA” refers to a RNA polynucleotide being or including the target sequence. In other words, the target RNA may be a RNA polynucleotide or a part of a RNA polynucleotide to which a part of the gRNA, i.e. the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex including CRISPR effector protein and a gRNA is to be directed. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

[0118] RNA-targeting effector proteins include CRISPR / Cas Class II enzymes. Examples include Type V and Type VI enzymes, such as those used with the SHERLOCK and DETECTR methods of nucleic acid detection. These include, but are not limited to, Casl4, Casl3a, Casl3b, and Casl2a, among others. In some aspects, the Cas protein can be activated by a nucleic acid derived from a specific virus. Accordingly, the probe can be used to detect the presence or absence of said virus. Examples of viruses which can be detected by the disclosed probes include, but are not limited to, influenza (e.g., H5N1), coronavirus (e.g., SARS-CoV-2), and measles.

[0119] In some aspects, the cleavage site can include a non-nucleic acid molecule, for example, an amino acid, a carbohydrate, a polyester, a plastic (e.g., PET or PLA), a polyhydroxyalkanoate, or a cleavable linker. It is considered that, when the cleavage site includes a polymeric compound (e.g., polyester, plastic, polyhydroxyalkanoate), the cleavage site may include any monomers, oligomers, or polymers of said polymeric compound. The term “linker” generally refers to a chemical moiety that is capable of covalently linking two or more oligonucleotides, in which at least one bond within the linker is capable of being cleaved, such that at least two oligonucleotides are no longer covalently linked to one another after bond cleavage. It will be appreciated that a provided linker may include a region that is non- cleavable, as long as the linker also includes at least one bond that is cleavable.

[0120] Examples of linkers include, but are not limited to, a peptide linker, P-glucuronide linker, hydrazone linker, pH cleavable linker, photocleavable linker, vibration sensitive linker, temperature sensitive linker, or disulfide linker. These modifications can allow for recognition of the probe by the activated catalytic agent.

[0121] In some aspect, the catalytic agent can include a protease, P-glucuronidase, cellulase, lyase (e.g., decarboxylase, dehydratase, or aldolase), amylase, lipase, cutinase, or PHA depolymerase. Attorney Docket No. 10046-616W01

[0122] In some aspects, the catalytic agent can include a change in pH, a change in temperature, exposure to light (e.g., UV light), or exposure to vibration. Accordingly, the probe can be used to identify or monitor environmental conditions of a particular sample.

[0123] Buffered Compositions

[0124] In some aspects, also disclosed herein is a composition including any of the disclosed probes and a buffer. This buffer can stabilize the fluorescent metal nanocluster and associated nucleic acid sequence. For example, the buffer can include about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM of sodium phosphate. The pH of the buffer with sodium phosphate can be about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,

[0125] 7.3, or 7.5. Preferably, the buffer including sodium phosphate is present in the composition at about 20 mM at a pH of about 6.6.

[0126] The buffer can further include Tris-HCl. This can be present at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mM in the buffer. The buffer with HC1 can be at a pH of about 7.0, 7. 1, 7.2, 7.3,

[0127] 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2. Preferably, the buffer including HC1 is present at about 10 mM Tris-HCl at a pH of about 7.6.

[0128] The buffer can further include ammonium acetate. The ammonium acetate can be present at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM. The pH of the buffer with ammonium acetate can be 6.5, 6.6, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, or 7.5. Preferably, the buffer including ammonium acetate is present at about 10 mM at a pH of about 7.0. This buffer can further include MgCh at about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mM, and can further include CaCh at about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mM CaCh. Preferably, the buffer can include MgCh at about 2.5 mM and CaCh at about 0.5 mM. One of skill in the art will appreciate that each nuclease will require slightly different ingredients in its buffer in order to maximize its enzyme activity.

[0129] Multiplexing

[0130] Multiple probes can be used simultaneously. For example, two or more probes can be used that have different metal nanocluster components, or different nucleic acid components, or both. These different probes can have different properties, such that differential detection of a target, or quantification of a target, is possible. For example, the probes can fluoresce differently upon binding of different target nucleic acid sequences. They can also have different modifications, which are discussed above. For instance, they can have the same or different catalytic agents capable of cleaving the probe. For example, two different probes can each have a different sequence which is recognized by different catalytic nucleic acid enzymes. Two different probes can also have the same or different cleavable linkers. One or more of the Attorney Docket No. 10046-616W01 multiplexed linkers can be cleavable by a nuclease, while another is cleavable by a catalytic nucleic acid.

[0131] A multiplex assay including these probes can have 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different probes which are used simultaneously. The probes can fluoresce differently upon binding of different target nucleic acids and cleavage of the probe by the catalytic nucleic acid enzyme.

[0132] Kits

[0133] Also disclosed herein are kits. These kits can include any of the probes discussed herein, as well as a buffer suitable for stabilizing the fluorescent metal nanocluster and associated nucleic acid sequence. These buffers are discussed above. The kit can include multiplexed probes, or single probes. Other components can also be included in the kit.

[0134] Methods

[0135] Disclosed herein are methods of using the probes and compositions thereof discussed herein. For example, disclosed herein is a method of detecting activity of a catalytic agent in a sample, the method including: a) providing any of the disclosed probes or compositions to the sample; and b) detecting the detectable signal, thereby detecting activity of the catalytic agent.

[0136] In some aspects, if the catalytic agent is activated by an analyte, the activity of the catalytic agent as indicated by the presence of the detectable signal can indicate the present of the analyte in the sample. In some aspects, if the catalytic agent is a change in an environmental condition (e.g., pH, temperature, exposure to light, exposure to vibration, etc.), the method can be used to identify and / or monitor environmental conditions of the sample.

[0137] In some aspects, the sample can be a cell, a tissue, a biological fluid, a cell lysate, an environmental sample, or an industrial sample. In some aspects, the method can take place in vivo, in vitro, or in situ.

[0138] EXAMPLES

[0139] Example 1: Biosensing Clozapine

[0140] Overview:

[0141] Antipsychotic medications are the main class of drugs to treat schizophrenia patients, reducing / relieving their symptoms of psychosis such as delusions and hallucinations. It is estimated that in the US the prevalence of schizophrenia and related psychotic disorders ranges between 0.25% and 0.64% of the population. Among all antipsychotic medications, clozapine is not only the most effective but also has a well-established relationship between its blood Attorney Docket No. 10046-616W01 concentration and its therapeutic effect. However, clozapine can cause serious side effects (e.g., seizures or neutropenia) when its blood concentration is higher than 1,300 ng / ml. Many clinicians are thus reluctant to commence clozapine treatment as they are concerned that patients may not comply with regular venous blood monitoring, making clozapine the most underutilized treatment for schizophrenia.

[0142] Several analytical methods have been developed for measuring the blood concentration of clozapine and its major metabolites at central laboratory settings. These include liquid chromatography (LC) with ultraviolet (UV) detection. LC with mass spectrometry detection and gas-liquid-chromatography with nitrogen selective detector. Among them, the most widely used method is the high-performance LC with mass spectrometry (LC-MS) due to its sensitivity, reliability and ease of assay automation. However, LC-MS requires samples to be shipped from the clinic to the central laboratory for analysis. Consequently, there is a significant delay of several days before the testing results become available to clinicians and patients. Therefore, there is an urgent need for a fast, affordable, reliable, and sensitive way to routinely monitor clozapine concentration in the blood at patients’ homes.

[0143] To monitor the drug effects of clozapine, neutrophil counts (PointCheck™ by Leuko Labs) and immunoassays (MyCare™ by Saladax Biomedical) are the two major tests in today’s market. However, neutrophil counts are an indirect way to estimate the clozapine level when clozapine -induced agranulocytosis becomes evident in the patients. Although MyCare™ immunoassay is a direct way to monitor clozapine concentration in patients’ blood, it suffers from the issues of poor limitation-of-detection (LoD, 208 nM), unstable readings (1.6%) and high assay cost ($14 per test). Disclosed herein is a new biosensing tool that is more sensitive, reliable and affordable than the MyCare assay.

[0144] Exploring the new properties of low-cost recognition molecules (i.e., aptamers and aptazymes) and nanomaterial probes (e.g., Subak probe) can expand the beneficiaries of point- of-care testing (POCT) sensors to resource-limited regions, avoiding deadly side effects due to overtreatment and poor therapeutic outcomes due to undertreatment. This project not only creates new clozapine sensors, but also establishes universal strategies for small-molecule detection.

[0145] Disclosed herein is a highly sensitive but affordable clozapine sensor based on DNAzymes. DNAzymes (i.e., DNA-based enzymes) have become an important molecular tool in today’s chemical measurements and imaging. Combined with fluorescence, colorimetry SERS, electrochemistry, and electrochemiluminescence detection schemes, DNAzyme sensors have reached a limit of detection (LoD) down to nM or even pM range. Attorney Docket No. 10046-616W01

[0146] Extended from the original metal ion sensors, DNAzymes can be integrated with aptamers, creating aptazymes for detecting small molecules such as adenosine, AMP, and ATP. The main advantage of DNAzymes and aptazymes is their signal amplification due to multiple enzymatic turnovers (i.e., one target molecule can thus generate multiple fluorescence cleavage signals). However, highly sensitive DNAzyme sensors all rely on expensive FRET substrates as reporters.

[0147] Current results: A low-cost non-FRET probe termed Subak has been developed that fluoresces differently upon nuclease cleavage (2024 Nature Nanotechnology). In short, a silver cluster, Agi39+, templated in a 37-nt long hairpin DNA exhibits green fluorescence emission. When the DNA backbone is cleaved by a nuclease such as DNase I and activated Casl2a, silver cluster fission takes place, which turns the green Agi39+cluster into a red Agio7+cluster and non-emissive Agi-3 fission fragments. This AgNC transformation process induced by an enzymatic reaction on DNA is extremely reliable, giving an emission peak red shift by 90 nm which can be easily observed by the naked eye. The red Agio7+cluster is found templated in two major DNA fragments, a 16-nt long and a 25 -nt long fragments, suggesting two preferred cutting sites by DNase I on this Agi39+hosted 37-nt hairpin DNA.

[0148] This phenomenon was coined Enzyme -Induced Fission of AgNCs (EIFiAg). Under UV excitation, a green (540 nm) to red (630 nm) color conversion was clearly seen when the Subak probe was fragmented by DNase I, which correlated well with the nuclease quantity and showed a clear isoemissive point in the emission spectra.

[0149] Compared to the widely used FRET reporters DNaseAlert in the DETECR assay, RNaseAlert in the SHERLOCK assay, and TaqMan in qPCR, the advantage of Subak’s ratiometric sensing was clearly seen in a higher fold change (15 times higher), with a comparable LoD (1.19 pM vs. 0.54 pM) and dynamic range ( 103) . In addition, Subak reporters can be easily prepared at room temperature in a single-pot reaction and have only one-sixtieth of the cost of DNaseAlert. As Subak relies on silver stoichiometry change (e.g., from green Agi3 to red Agio) as the signal transduction method, it does not require dual-labeling and can work even without any purification.

[0150] Disclosed herein is a clozapine aptazyme sensor (FIG. 1A). Results indicated that Subak can be used as a fluorescent substrate to monitor RNase (FIG. IB) and DNAzyme (FIG. 1C) activities. In the DNAzyme industry (e.g., the heavy metal detection company Alpha Measurement Solutions)' , the most expensive component for the DNAzyme assays is the FRET substrate. FRET substrate can be replaced by an ultralow-cost non-FRET probe, which creates a low-cost aptazyme sensor for monitoring clozapine’s blood concentration at patients’ homes. Attorney Docket No. 10046-616W01

[0151] The initial design of a new Subak probe that can be employed as a substrate for clozapine aptazyme detection has a uracil in the loop (FIG. 1A). Incorporating a uracil in the loop does not change the emission color of intact Subak. When this RNA-incorporated Subak probe is fragmented using an RNase A / Tl mix, which is known to cleave a uracil-containing substrate, the solution does show a red emission peak (FIG. IB). In an attempt to cut this RNA- incorporated Subak using a 10-23 DNAzyme 9129), a clear green color suppression and a new read peak (around 630 nm) were observed.

[0152] Probe design strategies are used to create new Subak probes specifically for DNAzyme detection.

[0153] Instead of going through the Agi3 to Agio stoichiometry change found in the DNase I fragmentation experiment, different Ag stoichiometry changes may exist in the DNAzyme - cleavage experiment. This is due to the fact that the preferred cleavage sites in the current Subak by DNase I are at T16 / T17 and A25 / C26 locations in the stem, while the designated RNA cutting site in the RNA-containing Subak is in the loop (FIG. 1A). It appears that by incorporating RNA at different locations in Subak probe, a variety of AgNC fission results can be attained (e.g., from Agi3 to Ags), thus generating more color conversion pairs among Subak probes.

[0154] Example 2: Subak Probes

[0155] A Subak probe with a hairpin DNA template can be used with the methods and systems described herein. FIG. 9 provides a schematic overview of the various modifications and uses of the Subak probes.

[0156] Example 3: Additional Experimental Data

[0157] Probes validated under various conditions (e.g., buffers, temperature, target strands): A study was conducted to characterize the DNA-templated silver nanocluster (DNA- AgNC) probes, Subak and its RNA derivative rSubak, under a broad spectrum of experimental conditions to evaluate their compatibility and robustness across diverse nucleic acid processing environments (FIGS. 10A-10C). This includes extensive testing in buffer systems commonly employed in nucleic acid workflows - Sodium Phosphate Buffer (pH 7.4), DNase I reaction buffer, rCutSmart buffer, and ammonium acetate buffer (the latter specifically for ESI-MS analysis). Incubation studies at both ambient (room temperature) and physiological temperatures (37°C) further provide insights into probe behavior under biologically relevant conditions.

[0158] These findings indicate that these probes exhibit substantial tolerance to a range of salts and divalent cations typically encountered in enzymatic reactions, including up to 50 mM Attorney Docket No. 10046-616W01 potassium acetate, 20 mM Tris-acetate (pH 7.9), 10 mM magnesium acetate, 2.5 mM magnesium chloride, and 0.5 mM calcium chloride, as well as the presence of lOO pg / mL recombinant albumin. These results suggest a reasonable level of chemical stability.

[0159] Initial thermal stability assessments revealed that while Subak-1 and Subak-2 maintain their core functionality, exposure to PCR-relevant thermal cycling (e.g., 95 °C denaturation and 60°C annealing / extension) results in a marked reduction of green fluorescence after approximately 5-10 cycles (FIG. HA). Notably, this decrease in green channel intensity concurrently enhances the ratiometric signal (1630 / 1540), producing a sharper contrast during the green-to-red fluorescence transition (FIG. 11B). This emergent property reinforces the probe’s potential utility in digestion-based workflows and endpoint analyses. Nevertheless, additional molecular engineering is required to further stabilize the probe under prolonged thermal cycling, particularly for integration into quantitative PCR workflows.

[0160] Supporting data in FIGS. 10A-10C and FIGS. 11A-11B highlight ratiometric fluorescence changes (I625 / I530), isoemissive behavior during RNase digestion, and ESI-MS spectral validation of probe integrity under varied buffer conditions.

[0161] Probes optimized in various RT qPCR and CRISPR assays:

[0162] Reagents for RT qPCR assays'. Another study performed initial thermal stability testing of Subak probes under PCR-relevant conditions to evaluate their suitability for real-time RT- qPCR applications. While Subak retains its ability to detect nucleic acids, green fluorescence exhibited a marked decrease after 5-10 thermal cycles (FIG. HA). Importantly, analysis of ratiometric sensing (1630 / 1540) showed that signal transitions remain comparable despite this reduction, supporting the probe’s continued utility in qPCR assay workflows (FIG. 11B).

[0163] Reagents for CRISPR assays: Significant progress has been achieved in adapting rSubak for CRISPR-Casl3a applications, specifically SHERLOCK assays. Iterative probe redesign, including targeted ribonucleotide substitutions (positions T16-T21), yielded constructs with robust and reproducible green-to-red fluorescence conversion upon Casl Sa- mediated collateral cleavage. Sensitivity benchmarking established a limit of detection (LoD) in the 5-10 pM range - comparable to the performance of commercial RNase Alert probes - thus validating rSubak’s suitability for nucleic acid detection in diagnostic workflows.

[0164] Supporting data (FIG. 12, FIGS. 13A-13C, and TABLE 2) present detailed fluorescence kinetics and endpoint analyses of influenza A RNA targets, demonstrating robust and consistent ratiometric outputs with improved assay readability. Additional evaluations with other RNA viruses, including SARS-CoV-2 and measles virus, further confirmed the Attorney Docket No. 10046-616W01 successful green-to-red signal transition across diverse targets, underscoring the broad applicability of rSubak in CRISPR-based detection workflows.

[0165] TABLE 2. Summary table comparing the analytical performance of rSubak (L525), and ratiometric (I625 / I530) with RNaseAlert (1520). rSubak achieves markedly lower limits of detection (LoD) across all three viral targets, does not require post-synthesis purification, offers ratiometric readout, and reduces probe cost from ~$75.60 / nmol (RNaseAlert) to ~$l / nmol.

[0166] Probe’s reliability and performance comparisons with commercial products: To assess practical utility, rSubak was benchmarked against RNaseAlert in Casl3a SHERLOCK assays targeting SARS-CoV-2, H5N1, and measles virus RNA. rSubak demonstrated comparable sensitivity while offering an enhanced fluorescence fold-change, attributed to its unique ratiometric design. This dual-channel output facilitates more reliable detection by mitigating environmental and instrumental variability, a key advantage over single-channel probes. Additionally, rSubak synthesis was achieved at approximately 1.5% of the RNaseAlert production cost, underscoring its potential for scalable, cost-effective deployment in resourcelimited settings.

[0167] This project demonstrated that DNA-RNA hybrid silver nanocluster reporters (rSubak) are viable non-FRET alternatives for nucleic acid detection with unique colorimetric switching. The green-to-red fluorescence conversion and ratiometric output provide quantification advantages, while maintaining low-cost synthesis and simplified design. These outcomes support clear advantages over current FRET-based probes.

[0168] EXAMPLE ASPECTS

[0169] Example 1: A composition comprising: a) a probe, wherein the probe comprises a nucleic acid sequence and a fluorescent metal nanocluster associated with the nucleic acid Attorney Docket No. 10046-616W01 sequence; and b) a catalytic agent capable of cleaving the probe, wherein the agent is not a nuclease.

[0170] Example 2: The composition of any examples herein, particularly Example 1, wherein the catalytic agent is capable of interacting with an analyte, wherein said analyte can activate the catalytic agent so that it is capable of cleaving the probe.

[0171] Example 3: The composition of any examples herein, particularly Example 2, wherein the analyte comprises nucleic acid, a small molecule, a protein, or a metal ion.

[0172] Example 4: The composition of any examples herein, particularly Example 2 or Example 3, wherein, in the presence of the analyte, the catalytic agent is activated to cleave the probe.

[0173] Example 5: The composition of any examples herein, particularly Examples 1-4, wherein the nucleic acid sequence of the probe comprises at least one RNA nucleotide.

[0174] Example 6: The composition of any examples herein, particularly Examples 1-5, wherein the probe further comprises at least one molecule which is not a nucleic acid.

[0175] Example 7: The composition of any examples herein, particularly Example 6, wherein the molecule comprises at least one amino acid or a cleavable linker.

[0176] Example 8: The composition of any examples herein, particularly Example 7, wherein the cleavable linker comprises a peptide linker, P-glucuronide linker, hydrazone linker, pH cleavable linker, photocleavable linker, vibration sensitive linker, temperature sensitive linker, or disulfide linker.

[0177] Example 9: The composition of any of any examples herein, particularly Examples 1- 8, wherein the catalytic agent comprises a protease, P-glucuronidase, cellulase, lyase, amylase, lipase, cutinase, PHA depolymerase, or a DNAzyme.

[0178] Example 10: The composition of any examples herein, particularly Example 9, wherein the DNAzyme cleaves RNA or DNA.

[0179] Example 11: The composition of any examples herein, particularly Example 9 or Example 10, wherein the DNAzyme is an aptazyme which is sensitive to an analyte.

[0180] Example 12: The composition of any examples herein, particularly Example 9, wherein the lyase comprises decarboxylase, dehydratase, or aldolase.

[0181] Example 13: The composition of any examples herein, particularly Examples 1-12, wherein the composition further comprises a metal ion cofactor.

[0182] Example 14: The composition of any examples herein, particularly Example 13, wherein the metal ion cofactor comprises at least one of Pb2+, Cu+, Cu2+, UCh2+, Zn2+, Mg2+, Hg2+, Ag+, Ca2+, Tl3+, Cd2+, C+, Co2+, Ni2+, Na+, Li+, Fe2+, Fe3+, and Mn2+. Attorney Docket No. 10046-616W01

[0183] Example 15: The composition of any examples herein, particularly Examples 1-14, wherein the composition further comprises a buffer that stabilizes the fluorescent metal nanocluster and associated nucleic acid sequence.

[0184] Example 16: The composition of any examples herein, particularly Example 5, wherein a single RNA nucleotide is present in the probe.

[0185] Example 17: The composition of any examples herein, particularly Example 5, wherein more than one RNA molecule is present in the probe.

[0186] Example 18: The composition of any examples herein, particularly Examples 1-17, wherein the nucleic acid sequence of the full probe is 3-150 nucleotides in length.

[0187] Example 19: The composition of any examples herein, particularly Examples 1-18, wherein the nucleic acid sequence of the probe can comprise a secondary structure.

[0188] Example 20: The composition of any examples herein, particularly Example 19, wherein said secondary structure comprises a stem-loop (hairpin) structure.

[0189] Example 21: The composition of any examples herein, particularly Examples 1-20, wherein the fluorescent metal nanocluster is a silver nanocluster or a silver-containing nanocluster.

[0190] Example 22: The composition of any examples herein, particularly Examples 1-21, wherein the probe is a modified Subak probe.

[0191] Example 23: The composition of any examples herein, particularly Example 22, wherein the probe is selected from the group comprising SEQ ID NO: 2-1518.

[0192] Example 24: A probe comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises at least one RNA nucleotide, and a fluorescent metal nanocluster associated with the nucleic acid sequence.

[0193] Example 25: The probe of any examples herein, particularly Example 24, wherein the probe comprises only one RNA nucleotide.

[0194] Example 26: The probe of any examples herein, particularly Example 24, wherein the probe comprises more than one RNA nucleotide.

[0195] Example 27: The probe of any examples herein, particularly Examples 24-26, wherein the nucleic acid of the probe is 6 - 100 nucleotides in length.

[0196] Example 28: The probe of any examples herein, particularly Examples 24-27, wherein the nucleic acid sequence of the probe comprises a secondary structure.

[0197] Example 29: The probe of any examples herein, particularly Example 28, wherein the secondary structure is a stem -loop structure. Attorney Docket No. 10046-616W01

[0198] Example 30: A probe comprising: a) a nucleic acid sequence; b) a non-nucleic acid molecule; and c) a fluorescent metal nanocluster associated with the nucleic acid sequence.

[0199] Example 31: The probe of any examples herein, particularly Example 30, wherein the non-nucleic acid molecule comprises an amino acid, a carbohydrate, a polyester, a plastic, a polyhydroxyalkanoate, or a cleavable linker.

[0200] Example 32: The probe of any examples herein, particularly Example 31, wherein the cleavable linker comprises a peptide linker, P-glucuronide linker, hydrazone linker, pH cleavable linker, photocleavable linker, vibration sensitive linker, temperature sensitive linker, or disulfide linker.

[0201] Example 33: The probe of any examples herein, particularly Examples 30-32, wherein the probe comprises only one cleavable linker.

[0202] Example 34: The probe of any examples herein, particularly Examples 30-32, wherein the probe comprises more than one cleavable linker.

[0203] Example 35: The probe of any examples herein, particularly Examples 30-34, wherein the nucleic acid of the probe is 6-100 nucleotides in length.

[0204] Example 36: The probe of any examples herein, particularly Example 35, wherein the nucleic acid sequence of the probe comprises a secondary structure.

[0205] Example 37: The probe of any examples herein, particularly Example 36, wherein the secondary structure is a stem -loop structure.

[0206] Example 38: A kit comprising the probe of any examples herein, particularly Examples 24-37, and a buffer suitable for stabilizing the fluorescent metal nanocluster and associated nucleic acid sequence.

[0207] Example 39: The kit of any examples herein, particularly Example 38, wherein the buffer comprises about 10 mM of ammonium acetate and about 20 mM sodium phosphate buffer.

[0208] Example 40: The kit of any examples herein, particularly Example 38 or Example 39, wherein the kit further comprises a catalytic enzyme.

[0209] Example 41: The kit of any examples herein, particularly Example 40, wherein the catalytic enzyme comprises a protease, P-glucuronidase, cellulase, lyase, amylase, lipase, cutinase, PHA depolymerase, or a DNAzyme.

[0210] Example 42: The kit of any examples herein, particularly Example 41, wherein the DNAzyme cleaves RNA or DNA.

[0211] Example 43: The kit of any examples herein, particularly Example 42, wherein the DNAzyme is an aptazyme. Attorney Docket No. 10046-616W01

[0212] Example 44: The kit of any examples herein, particularly Example 43, wherein the kit further comprises a substance which can cleave a cleavable linker.

[0213] Example 45: A composition comprising: a) at least two probes, wherein each probe comprises a nucleic acid sequence and a fluorescent metal nanocluster associated with the nucleic acid sequence, wherein the probes fluoresce differently upon binding of different target nucleic acid sequences; and b) at least one catalytic agent capable of cleaving at least one of the probes.

[0214] Example 46: The composition of any examples herein, particularly Example 45, wherein the at least one catalytic agent comprises a protease, P-glucuronidase, cellulase, lyase, amylase, lipase, cutinase, PHA depolymerase, or a DNAzyme.

[0215] Example 47: The composition of any examples herein, particularly Example 46, wherein the DNAzyme cleaves RNA or DNA.

[0216] Example 48: The composition of any examples herein, particularly Example 47, wherein the DNAzyme is an aptazyme.

[0217] Example 49: The composition of any examples herein, particularly Examples 45-48, wherein the composition can detect an analyte.

[0218] Example 50: The composition of any examples herein, particularly Example 49, wherein the analyte comprises nucleic acid, a small molecule, a protein, or a metal ion.

[0219] Example 51: The composition of any examples herein, particularly Example 49 or Example 50, wherein, in the presence of the analyte, the catalytic agent is activated to cleave the probe.

[0220] Example 52: The composition of any examples herein, particularly Examples 45-51, wherein the nucleic acid sequence of at least one probe comprises at least one RNA nucleotide.

[0221] Example 53: The composition of any examples herein, particularly Examples 45-51, wherein the probe further comprises at least one molecule which is not a nucleic acid.

[0222] Example 54: The composition of any examples herein, particularly Example 53, wherein the molecule comprises at least one amino acid or a cleavable linker.

[0223] Example 55: The composition of any examples herein, particularly Example 54, wherein the cleavable linker comprises a peptide linker, P-glucuronide linker, hydrazone linker, pH cleavable linker, photocleavable linker, vibration sensitive linker, temperature sensitive linker, or disulfide linker.

[0224] Example 56: The composition of any examples herein, particularly Examples 45-55, wherein the probes fluoresce differently upon binding of different analytes and cleavage of the probe by the catalytic agent. Attorney Docket No. 10046-616W01

[0225] Example 57: The composition of any examples herein, particularly Examples 45-56, wherein at least one of the probes is cleavable by a nuclease, and the other by catalytic nucleic acid enzyme.

[0226] Example 58: The composition of any examples herein, particularly Examples 45-57, wherein the composition comprises a buffer that stabilizes the fluorescent metal nanocluster and associated nucleic acid sequence.

[0227] Example 59: The composition of any examples herein, particularly Example 58, wherein the buffer further comprises phosphate and / or acetate in an amount sufficient to stabilize the fluorescent metal nanocluster and associated nucleic acid sequence.

[0228] Example 60: The composition of any examples herein, particularly Examples 45-59, wherein the nucleic acid of the probe is about 6-100 nucleotides in length.

[0229] Example 61: The composition of any examples herein, particularly Examples 45-60, wherein the probe can further comprise secondary structure.

[0230] Example 62: The composition of any examples herein, particularly Example 61, wherein said secondary structure comprises a stem-loop (hairpin) structure.

[0231] Example 63: A method of detecting presence of an analyte, the method comprising: a) contacting a sample containing the analyte with: i) at least one probe comprising a nucleic acid sequence and a fluorescent metal nanocluster associated with the nucleic acid sequence; and ii) a catalytic agent which is capable of cleaving the probe of step i) in the presence of the analyte; and b) measuring a detectable signal produced by cleavage of the probe by the catalytic agent, thereby detecting the analyte.

[0232] Example 64: The method of any examples herein, particularly Example 63, wherein the analyte comprises nucleic acid, a small molecule, a protein, or a metal ion.

[0233] Example 65: The method of any examples herein, particularly Examples 63 or Example 64, wherein the nucleic acid sequence of the probe comprises at least one RNA nucleotide.

[0234] Example 66: The method of any examples herein, particularly Example 63 or Example 64, wherein the probe further comprises at least one molecule which is not a nucleic acid.

[0235] Example 67: The method of any examples herein, particularly Example 66, wherein the molecule comprises at least one amino acid or a cleavable linker.

[0236] Example 68: The method of any examples herein, particularly Example 67, wherein the cleavable linker comprises a peptide linker, P-glucuronide linker, hydrazone linker, pH cleavable linker, photocleavable linker, vibration sensitive linker, temperature sensitive linker, or disulfide linker. Attorney Docket No. 10046-616W01

[0237] Example 69: The method of any of any examples herein, particularly Examples 64-68, wherein the catalytic agent comprises a protease, P-glucuronidase, cellulase, lyase, amylase, lipase, cutinase, PHA depolymerase, or a DNAzyme.

[0238] Example 70: The method of any examples herein, particularly Example 69, wherein the DNAzyme cleaves RNA or DNA.

[0239] Example 71: The method of any examples herein, particularly Example 69 or Example 70, wherein the DNAzyme is an aptazyme which is sensitive to an analyte.

[0240] Example 72: The method of any examples herein, particularly Example 69, wherein the lyase comprises decarboxylase, dehydratase, or aldolase.

[0241] Example 73: The method of any examples herein, particularly Example 64, wherein the metal ion comprises Pb2+, Cu+, Cu2+, UCh2+, Zn2+, Mg2+, Hg2+, Ag+, Ca2+, Tl3+, Cd2+, Cr3+, Co2+, Ni2+, Na+, Li+, Fe2+, Fe3+, and Mn2+.

[0242] Example 74: A probe comprising 90% identity or more to any one of SEQ ID NOS: 2- 18.

[0243] Example 75: A probe comprising: a nucleic acid sequence, wherein said nucleic acid sequence comprises a cleavage site comprising at least one RNA nucleotide and / or a non- nucleic acid molecule; and a fluorescent metal nanocluster associated with the nucleic acid sequence; wherein the fluorescent metal nanocluster produces a detectable signal upon cleavage of the nucleic acid sequence by a catalytic agent.

[0244] Example 76: The probe of any examples herein, particularly Example 75, wherein the cleavage site comprises from 1 to 6 RNA nucleotides.

[0245] Example 77: The probe of any examples herein, particularly Example 75, wherein the nucleic acid sequence comprises DNA and the cleavage site comprises at least one RNA nucleotide.

[0246] Example 78: The probe of any examples herein, particularly Example 75, wherein the cleavage site comprises at least one RNA nucleotide and the catalytic agent comprises a nuclease, a ribozyme, or a DNAzyme.

[0247] Example 79: The probe of any examples herein, particularly Example 78, wherein the nuclease is RNase or Casl3.

[0248] Example 80: The probe of any examples herein, particularly Example 79, wherein the Casl3 is activated by a viral nucleic acid.

[0249] Example 81: The probe of any examples herein, particularly Example 78, wherein the DNAzyme is an aptazyme which is activated by an analyte comprising a nucleic acid, a small molecule, a protein, or a metal ion. Attorney Docket No. 10046-616W01

[0250] Example 82: The probe of any examples herein, particularly Example 75, wherein the non-nucleic acid molecule comprises an amino acid, a carbohydrate, a polyester, a plastic, a polyhydroxyalkanoate, or a cleavable linker.

[0251] Example 83: The probe of any examples herein, particularly Example 82, wherein the cleavable linker comprises a peptide linker, P-glucuronide linker, hydrazone linker, pH cleavable linker, photocleavable linker, vibration sensitive linker, temperature sensitive linker, or disulfide linker.

[0252] Example 84: The probe of any examples herein, particularly Example 75, wherein the nucleic acid sequence comprises DNA and / or RNA and the cleavage site comprises a non- nucleic acid molecule.

[0253] Example 85: The probe of any examples herein, particularly Example 75, wherein the cleavage site comprises a non-nucleic acid molecule and the catalytic agent comprises a protease, P-glucuronidase, cellulase, lyase, amylase, lipase, cutinase, or PHA depolymerase.

[0254] Example 86: The probe of any examples herein, particularly Example 85, wherein the lyase comprises decarboxylase, dehydratase, or aldolase.

[0255] Example 87: The probe of any examples herein, particularly Example 75, wherein the cleavage site comprises a non-nucleic acid molecule and the catalytic agent comprises a change in pH, a change in temperature, exposure to light, or exposure to vibration.

[0256] Example 88: The probe of any examples herein, particularly Example 75, wherein the nucleic acid sequence comprises from about 3 to about 150 nucleotides.

[0257] Example 89: The probe of any examples herein, particularly Example 75, wherein the fluorescent metal nanocluster is a silver nanocluster or a silver-containing nanocluster.

[0258] Example 90: The probe of any examples herein, particularly Example 75, wherein the detectable signal is a change in color of the fluorescent metal nanocluster.

[0259] Example 91: The probe of any examples herein, particularly Example 75, wherein the probe comprises about 80% similarity or more to any one of SEQ ID NOS: 2-18.

[0260] Example 92: A composition comprising the probe of any examples herein, particularly Example 75, and a buffer that stabilizes the fluorescent metal nanocluster and associated nucleic acid sequence.

[0261] Example 93: A method of detecting activity of a catalytic agent in a sample, the method comprising: a) providing the probe of any examples herein, particularly Example 75 to the sample; and b) detecting the detectable signal, thereby detecting activity of the catalytic agent.

[0262] Example 94: The method of any examples herein, particularly Example 93, wherein the activity of the catalytic agent indicates presence of an analyte in the sample. Attorney Docket No. 10046-616W01

[0263] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.

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[0367] Attorney Docket No. 10046-616W01

[0368] SEQUENCES Attorney Docket No. 10046-616W01

Claims

Attorney Docket No. 10046-616W01WHAT IS CLAIMED IS:

1. A probe comprising: a nucleic acid sequence, wherein said nucleic acid sequence comprises a cleavage site comprising at least one RNA nucleotide and / or a non-nucleic acid molecule; and a fluorescent metal nanocluster associated with the nucleic acid sequence; wherein the fluorescent metal nanocluster produces a detectable signal upon cleavage of the nucleic acid sequence by a catalytic agent.

2. The probe of claim 1, wherein the cleavage site comprises from 1 to 6 RNA nucleotides.

3. The probe of claim 1, wherein the nucleic acid sequence comprises DNA and the cleavage site comprises at least one RNA nucleotide.

4. The probe of claim 1, wherein the cleavage site comprises at least one RNA nucleotide and the catalytic agent comprises a nuclease, a ribozyme, or a DNAzyme.

5. The probe of claim 4, wherein the nuclease is RNase or Casl3.

6. The probe of claim 5, wherein the Casl3 is activated by a viral nucleic acid.

7. The probe of claim 4, wherein the DNAzyme is an aptazyme which is activated by an analyte comprising a nucleic acid, a small molecule, a protein, or a metal ion.

8. The probe of claim 1, wherein the non-nucleic acid molecule comprises an amino acid, a carbohydrate, a polyester, a plastic, a polyhydroxyalkanoate, or a cleavable linker.

9. The probe of claim 8, wherein the cleavable linker comprises a peptide linker, - glucuronide linker, hydrazone linker, pH cleavable linker, photocleavable linker, vibration sensitive linker, temperature sensitive linker, or disulfide linker.

10. The probe of claim 1, wherein the nucleic acid sequence comprises DNA and / or RNA and the cleavage site comprises a non-nucleic acid molecule.Attorney Docket No. 10046-616W0111. The probe of claim 1, wherein the cleavage site comprises a non-nucleic acid molecule and the catalytic agent comprises a protease, P-glucuronidase, cellulase, lyase, amylase, lipase, cutinase, or PHA depolymerase.

12. The probe of claim 11, wherein the lyase comprises decarboxylase, dehydratase, or aldolase.

13. The probe of claim 1, wherein the cleavage site comprises a non-nucleic acid molecule and the catalytic agent comprises a change in pH, a change in temperature, exposure to light, or exposure to vibration.

14. The probe of claim 1, wherein the nucleic acid sequence comprises from about 3 to about 150 nucleotides.

15. The probe of claim 1, wherein the fluorescent metal nanocluster is a silver nanocluster or a silver-containing nanocluster.

16. The probe of claim 1, wherein the detectable signal is a change in color of the fluorescent metal nanocluster.

17. The probe of claim 1, wherein the probe comprises about 80% similarity or more to any one of SEQ ID NOS: 2-18.

18. A composition comprising the probe of claim 1 and a buffer that stabilizes the fluorescent metal nanocluster and associated nucleic acid sequence.

19. A method of detecting activity of a catalytic agent in a sample, the method comprising: a) providing the probe of claim 1 to the sample; and b) detecting the detectable signal, thereby detecting activity of the catalytic agent.

20. The method of claim 18, wherein the activity of the catalytic agent indicates presence of an analyte in the sample.

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