WO2025199140A1 - Methods and compositions related to dna-locked beacons for sensing of active enzymes - Google Patents

Abstract

Enzymes play an important role in many biological processes, and as such, are linked to numerous diseases including infectious diseases and cancer. There is now a new class of enzyme activity sensors called nucleic acid-locked beacons.

Description

METHODS AND COMPOSITIONS RELATED TO DNA-LOCKED BEACONS FOR

SENSING OF ACTIVE ENZYMES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/566,637, filed March 18, 2024, and U.S. Provisional Application No. 63/668,441, filed July 8, 2024, which are each incorporated by reference herein in their entireties.

REFERENCE TO SEQUENCE LISTING

[0002] The sequence listing submitted on March 18. 2025. as an .XML file entitled “10046- 607WOl_ST26.xml” created on March 12, 2025, and having a file size of 28,051 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

[0003] Enzymes play critical roles in nearly all biological processes from cell differentiation, apoptosis, protein turnover, to signal transduction. Consequently, the activity of enzymes is dysregulated in a variety of diseases, such as cancer, Alzheimer's, and atherosclerosis. By monitoring enzymes activity, diseases can be diagnosed and prognosed. Importantly, as enzymes are often expressed in an inactive form and turned on only in response to specific chemical cues, the abundance of enzymes does not always correlate with their activity. Current probes for monitoring active enzymes are limited by their sensitivity and multiplexing capabilities - it is challenging to detect low concentrations of active enzy mes in a scalable and multiplexed format.

[0004] Thus, there exists a need in the art to rapidly and easily detect the presence of active enzymes, especially at low concentrations. This need is satisfied by the present disclosure.

SUMMARY

[0005] Disclosed herein are DNA-locked beacons, also referred to as DNA-locked peptide beacons (DLPBs) or cleavable, locked initiator probes (CLIPs), as an alternative to commercial probes in these aspects, enhancing the detection of active enzy mes (e.g., active proteases). DNA-barcoded strategies, wherein a peptide or other cleavable molecule is attached to a unique DNA barcode can alleviate some of the challenges associated with commercial probes. These constructs, by pairing the enzy me-specific sensing capabilities of the cleavable molecule with nucleic acids, are capable of detecting active enzymes with remarkable sensitivity, selectivity', and with the advantage of barcoding. In existing systems, a separation step is necessary to separate intact DNA-barcoded peptides from those digested by the enzyme. This additional step can add complexity and also may result in losses. Moreover, in systems involving attachment of peptides to nanoparticle (NP) surfaces, longer peptides are often necessary as shorter peptides are often not sterically accessible by the enzymes.

[0006] In an aspect, provided is a nucleic acid-locked beacon, wherein said nucleic acid- locked beacon includes: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence. [0007] In another aspect, provided is a composition including a nucleic acid-locked beacon, wherein said nucleic acid-locked beacon includes: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence.

[0008] In yet another aspect, provided is a composition including two or more nucleic acid- locked beacons, wherein each of said nucleic acid-locked beacon includes: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein the two or more nucleic acid- locked beacons include different peptide sequences.

[0009] In yet another aspect, provided is a method of detecting the presence of an enzyme, the method comprising using any of the disclosed compositions to detect the presence of an enzyme.

[0010] In some aspects, the enzyme can be a marker of cancer, inflammation, infection, virus, cardiovascular disease, or other disease or disorder.

[0011] In yet another aspect, provided is a method of determining usefulness of a test compound in reducing or eliminating effects of an enzyme , the method including exposing any of the disclosed beacons to a test compound and determining effect of the test compound on action of the enzy me .

[0012] In yet another aspect, provided is a diagnostic assay for determining presence of an enzyme, the assay including any of the disclosed beacons.

[0013] In yet another aspect, provided is a method of treating and/or preventing a disease in a subject, the method comprising administering any of the disclosed compositions to the subject, wherein the signaling nucleic acid sequence can be configured to induce a therapeutic response when the composition is in the presence of an enzyme. [0014] Other systems, methods, features and/or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and/or advantages be included within this description and be protected by the accompanying claims.

BRIEF DESCRIPTION OF DRAWINGS

[0015] FIGURE 1A depicts treatment of DLPBs with a target protease results in the proteolytic cleavage of the peptide substrate. This cleavage leads to the spontaneous melting of the locked hairpin structure, which releases an unlocked signaling DNA handle for amplification and signal transduction. FIGURE IB depicts that, when combined with a CRISPR-Casl2a detection platform, the unlocked signaling DNA causes the generation of an amplified fluorescence response.

[0016] FIGURE 2A depicts MALDI-MS analysis of DLPBs and protease-treated DLPBs. After protease treatment, the peak at 12,569 m/z diminishes, and two new fragments, correlating with the expected proteolytic fragments of DLPBs, appear. FIGURE 2B depicts melting temperature of fluorophore-quencher (FQ) 3CL DLPBs which reveals that as- synthesized DLPBs form the hairpin structure, and after protease treatment are cleaved into fragments with a low er T_m. FIGURE 2C depicts CRISPR-mediated detection of 3CL DLPBs with and without a 30-minute incubation with 500 nM 3CL protease. FIGURE 2D is a comparison of 3CL DLPBs, FQ 3CL DLPBs, and commercial CTSB reporter upon incubation with 250 nM and 500 nM 3CL protease.

[0017] FIGURE 3A depicts proteases of interest - 3CL, CASP3, MMP7, and CTSB - and their corresponding peptide substrates that are incorporated into DLPBs. FIGURE 3B depicts activation of protease specific DLPBs after treatment with 500 nM CASP3, 250 nM MMP7, and 20 nM CTSB.

[0018] FIGURE 4A depicts CRISPR mediated signal response when CTSB DLPBs are incubated for 30 minutes with increasing concentrations of CTSB Protease. FIGURE 4B depicts a CTSB DLPB response at a fixed time point from incubation with increasing CTSB concentrations. Limit of Detection (LOD) analysis demonstrates an LOD of 150 pM for CTSB after 60 minutes. FIGURE 4C depicts a specificity assay for CTSB DLPBs when incubated with 20 nM non-specific proteases and proteins relative to untreated DLPBs.

[0019] FIGURES 5A-5D depict activation of CTSB DLPBs when treated with cell lysates from lines, HT29 (FIG. 5A), NCI-H508 (FIG. 5B). RKO (FIG. 5C). and SW-620 (FIG. 5D). FIGURES 5E-5F depict treatment of a commercial CTSB fluorogenic probe (FIG. 5E) and CTSB DLPBs (FIG. 5F) with SW-620 cell line and CTSB inhibitor.

[0020] FIGURE 6A depicts a modeled 30-8 DNA hairpin using Integrated Technologies Oligo Analyzer tool. FIGURE 6B depicts a modeled 10-8 DNA hairpin using Integrated Technologies OligoAnalyzer tool. FIGURE 6C depicts a modeled DNA non-hairpin 12-0 using Integrated Technologies OligoAnalyzer tool. A 2-base region in the activating sequence does show intramolecular hybridization. However, there is no end-to-end hairpining, and therefore this serves as a non-hairpining control. FIGURE 6D depicts a 3D model of 3CL DLPBs with activator DNA (red), peptide (cyan), linker (yellow), and 8-nt blocking DNA (blue). FIGURE 6E depicts a comparison of DLPBs to CRISPR-Casl2a enzymes. CRISPR- Casl2a modeled from protein data bank entry 6I1L.

[0021] FIGURES 7A-7B depict a hypothesized interaction between CRISPR-Casl2a enzymes and DLPBs (FIG. 7A) and protease-cleaved DLPBs (FIG. 7B). The conformation of DLPBs should restrict the activation of CRISPR-Casl2a due to both having a blocking sequence that restricts the hybridization of gRNA and a steric hindrance between the peptide loop into accessing the Casl2a enzyme. After DLPBs are cleaved by target proteases, the resultant single stranded activator DNA should have neither of these issues and activate CRISPR-Casl2a less impeded.

[0022] FIGURES 8A-8B depict temperature dependent fluorescence of Purchased Hairpin 12-8 and Non-hairpin 12-0 DNA strands in 50 mM NaCl (FIG. 8A) and CRISPR buffer (FIG. SB), with SYBR Green monitored via qPCR. It’s observed that the non-hairpining control melts at lower temperatures than the Hairpin 12-8 control, indicating that these strands have secondary structures as predicted. FIGURE 8C depicts melting temperature analysis of Cy5 and BHQ strand mixture, impure FQ 3CL DLPB, and pure FQ 3CL DLPB. Upon conjugation, the melting temperature of the FQ 3CL DLPB drastically increases in relation to the Cy5 and BHQ strand mixture. The impure FQ 3CL DLPB also shows an amount of low melting temperature impurities, which disappear after purification as seen in the pure FQ 3CL DLPB curve. FIGURE 8D depicts melting temperature analysis of Cy5 and BHQ strand mixture, and FQ 3CL DLPB in CRISPR buffer. The melting temperature of the FQ 3CL DLPB drastically increases in relation to the Cy5 and BHQ strand mixture, indicating a hairpin formation in the CRISPR buffer, supporting the hypothesis that DLPB structures, while hairpinned, are restricted from activating CRISPR.

[0023] FIGURE 9A depicts a PAGE gel containing Invitrogen™ Trackit™ Ultra Low Range DNA Ladder (lane A), ssDNA activator (lane B), 3CL pDNA conjugates (lane C), 3CL pDNA linker (lane D), impure DLPBs (lane E), and purified DLPBs (lane F). All samples are loaded at an approximate concentration of 1 nmol. Note that pDNA linker species show weaker fluorescence values than other species which has been previously seen. Distinct m/z increases are seen with each step in the synthesis of DLPBs indicating successful conjugation. Between impure and purified DLPBs species correlating to activator DNA, Peptide-DNA conjugates, and Peptide-DNA linkers, are all seen to disappear. Multiple bands are seen for pDNA linkers possibly due to the conformation of the DLPBs. FIGURE 9B depicts PAGE gel purification of an impure mixture of FQ 3CL DLPBs, unreacted peptide-DNA quencher and fluorophore labeled blocking strand. Loading was approximately 10 nM of the desired DLPB. The gel was observed by eye and underwent purification to isolate the indicated DLPB band. Note color of each band can be used as approximation for which species it correlates to, with the lower light blue bands correlating to Cy5 dye, the dark purple middle bands correlating to the BHQ, and the topmost royal blue bands seem like a mixture of these colors indicating that the species present contains both Cy5 and BHQ dyes. Also note that multiple bands also appear on this gel for each species possibly due to trace differences in either conformation or hybridization of the species.

[0024] FIGURE 10 depicts time dependent fluorescence assay upon incubation of 3CL FQ DLPBs with 3CL protease. The florescence enhancement over time indicates that the 3CL FQ DLPBs are being cleaved and dissociating as hypothesized.

[0025] FIGURE HA depicts a time dependent florescence assay showing the signal response from treating lOnM ribonucleoprotein complex (RNP) with varied concentration of ssDNA activator as seen in Table T3. Concentrations above 1 nM show larger standard deviation in triplicate and odd trends while concentrations at or below 0. 1 nM show minimal signal. To balance both a need for high signal, high repeatability, and to use fewer reagents a concentration of InM DNA activator was chosen as optimal. FIGURE 11B depicts a time dependent florescence assay showing signal response from treating 1 nM activator DNA with varied concentration of RNP. RNP background is from lOnM RNP complex. Concentrations of RNP below InM show incomplete saturation within 60 minutes while concentrations around lOnM saturate within the time frame. An RNP concentration of lOnM was chosen due to its ability to saturate within one hour with 1 nM of DNA activator. FIGURE 11C depicts a time dependent fluorescence assay of Activator DNA before and after treatment with protease. Upon incubating DNA only systems with protease no change is observed in CRISPR-Casl2a assays. In doing so there was no change seen with the activation of the DNA activator with and without protease incubation. This implies that certain proteases have no effect on the ability of CRISPR Casl2a signaling or that they are able to interfere with the binding of activator DNA with RNP complexes. FIGURES 11D-11E depict a time-dependent CRISPR assay of Activator DNA with and without the addition of a reverse complement (FIG. 11D) and a partial complement (FIG. HE). The Activator DNA appears to be significantly restricted when the full complement is added yet show s no restriction with the partial complement. This suggests that CRISPR cannot access fully double-stranded sequences, while, with partial complements, CRISPR-Casl2a can still access the activating sequence.

[0026] FIGURES 12A-12C depict activation of CRISPR-Casl2a with ssDNA activator DNA (FIG. 12A), activator DNA treated with the 16-nt blocking DNA (FIG. 12B), and activator DNA treated with the 24-nt blocking DNA (FIG. 12C). Activator DNA with and without treatment of the 16-nt blocking DNA is able to activate CRISPR-Casl2a with no impediment. However, when Activator DNA is treated with the 24-nt Blocking DNA heavy restriction on CRISPR-Casl2a activation is observed.

[0027] FIGURES 13A-13B depict enhancement of the 30-8 Hairpin with and without the addition of 24-nt blocking DNA (FIG. 13A) and 16-nt blocking DNA (FIG. 13B). The data displayed represents the average of two measurements, with error bars indicating the deviation between them. FIGURE 13C depicts that enhancement was obser ed at 60 minutes in DLPBs when combined with blocking strand C16 and 3CL protease. A negligible difference w as noted in the background activation of DLPBs, regardless of the presence of the C 16 blocking strand. [0028] FIGURE 14A-14B depict hypothesized activation of CRISPR-Casl2a via DNA hairpins. FIG. 14A shows that non-hairpin impurities or DNA hairpins may slowly activate Cast 2a enzymes. FIG. 14B shows that active C as 12a enzymes cleave the loop region of DNA hairpins causing them to degrade into new activating DNA causing an amplification cycle.

[0029] FIGURE 15 depicts hypothesized cleavage of DLPBs by CRISPR-Casl2a enzymes. Activated CRISPR-Casl2a enzymes have the ability to cleave ssDNA, but in DLPBs, the only region of ssDNA is the activating sequence for CRISPR-Casl2a. Therefore, cleavage within this region produces fragments of the activating DNA, rendering them inactive and unable to further activate CRISPR-Casl2a.

[0030] FIGURES 16A-16B depict time-dependent CRISPR-Casl2a florescence assays upon incubating 1 nM of impure (FIG. 16A) and purified (FIG. 16B) 3CL DLPBs with protease. Impure DLPBs show high background, likely due to the presence of non-hairpining unreacted species, while after HPLC purification, DLPB mixtures show retention of the same signal seen in impure samples but with the decrease of signal in control DLPB backgrounds. FIGURES 16C-16F depict signal increase upon incubating 10 nM, 1 nM, 0.1 nM, and 0.01 nM, of 3CL DLPB with 3CL protease. Minimal signal differences are gained using DLPB concentrations below 0. 1 nM. 1 nM DLPBs were chosen as an optimal concatenation and used for subsequent experiments. FIGURES 16G-16H depict detection of 2 pM 3CL protease via fluorescence-based CRISPR-CAS12A assay of 1 nM 3CL DLPB incubated at 25°C (FIG. 16G) and 37°C (FIG. 16H). Heating increases the background signal of DLPBs without protease treatment while there is no discernable difference in signal between DLPBs incubated with protease. FIGURES 16I-16J depict the limit of detection of 3CL via CRISPR-Casl2a assays with a 3CL specific DLPB. FIG. 161 depicts a time dependent florescence response from 3CL DLPBs incubated with 125-500 nM of 3CL protease for 30 minutes. FIG. 16J depicts a linear fit of response at 30 minutes. Calculation of the LOD via the 3*Sy/m method yields a LOD of 115 nM for 3CL. FIGURE 16K depicts the response upon incubating 3CL DLPBs with 500 nM and 1000 nM 3CL protease using a modified one pot procedure. Increases in response after protease incubation indicate one-pot detection with DLPBs is possible with minor modifications.

[0031] FIGURE 17 depicts the response upon incubating 1 nM and 100 nM commercial 3CL fluorogenic substrate. 100 nM FQ 3CL DLPB, and 1 nM DLPB with 250 nM and 500 nM 3CL protease.

[0032] FIGURE 18A depicts the response upon incubating 3CL DLPBs with 1 pM 3CL protease in the complex media of NCI-H508 cellular lysates. FIGURES 18B-18E depict treatment of 3CL, MMP7, CASP3, and CTSB DLPBs with cell lysates from lines NCI-H508, HT29, RKO, and SW-620. FIGURE 18F depicts the experimental LOD of SW-620 colon cancer cell lysates using CTSB DLPBs. FIGURE 18G depicts treatment of 100 nM of a CTSB commercial substrate with 15 pL of HT29, NCI-H508, RKO, and SW-620 line (~2million cells/mL). FIGURES 18H-18I depict treatment of commercial CTSB fluorogenic substrates with SW-620 cell lysates. Cell lysates were incubated with 1 nM (FIG. 18H) and 100 nM (FIG. 181) of the fluorogenic substrate, with and without the addition of a commercial CTSB inhibitor. FIGURE 18J depicts treatment of 3CL and CTSB DLPBs with SW-620 cell lysates. [0033] FIGURE 19A depicts protease dependent DNA hybridization with DLPBs. FIGURE 19B depicts protease triggered amplification and signaling DNA synthesis.

[0034] FIGURE 20 depicts structural modifications to peptide-DNA hairpins.

[0035] FIGURE 21 depicts various aspects of the beacon.

[0036] FIGURE 22A shows a cartoon structure of a CLIP including an initiator DNA, peptide, and blocking DNA. FIGURE 22B shows a molecular model of 3CL-CLIP with an idealized circular loop. FIGURE 22C is 3CL-CLIP showing peptide (SEQ ID NO: 1) and DNA (SEQ ID NO: 10) sequences.

[0037] FIGURE 23A shows the structure of a 40-nt gRNA hybridized to a 20-nt DNA initiator. FIGURE 23B shows a cartoon representation of the structure of the gRNA-DNA hybrid bound to CRISPR-Casl2a enzy me. FIGURE 23C shows a surface representation of the structure of the gRNA-DNA hybrid bound to CRISPR-Casl2a enzyme. These structures are modeled using protein data bank entry 611 L. FIGURE 23D shows the molecular structure of 3CL-CLIP.

[0038] FIGURE 24 depicts a scheme for synthesizing CLIPs.

[0039] FIGURE 25A shows the UV-Vis spectra of Initiator DNA. DNA's characteristic peak at 260 nm as well as the DBCOs characteristic 310 nm peak. FIGURE 25B shows initiator DNA treated with increasing concentrations of azide pep-3CL after 24 h. From the conjugation of DBCO to azide groups, the 310 peak decreases indicating successful conjugation of peptide-DNA conjugates. Treatment of 0:1, 1: 1, 3: 1, and 5: 1, azide to DBCO was measured. In samples of 3: 1 and 5: 1 equivalents the 310 nm peak appears to have disappeared indicating a successful complete conjugation.

[0040] FIGURE 26 shows the UV-Vis spectra of peptide-DNA linker before (blue) and after (red) overnight incubation with blocking strand DNA. Upon treatment the DBCO’s characteristic absorbance at 310 nm decreases approx. 18%, indicating that the azide on the peptide-DNA linker has successfully been attached.

[0041] FIGURE 27A shows a native PAGE gel containing (1) InvitrogenTM TrackitTM Ultra Low Range DNA Ladder, (2) ssDNA activator, (3) 3 CL peptide-DNA conjugates, (4) 3CL peptide-DNA-linker conjugates, (5) impure CLIPs, and (6) purified CLIPs. All samples are loaded in approximately 10 pmol amounts. The mobility of the structures gradually decreases with the addition of each component, indicating successful conjugation. Between impure and purified CLIPs species correlating to Initiator DNA, peptide-DNA conjugates, and peptide-DNA-linker conjugates, are all seen to disappear. Also note that multiple bands also appear on this gel for CLIPs possibly due to differences in either conformation or hybridization of the species. Lane 4 corresponds to the DNA-Peptide-Linker species which appears lighter than other species even at the same concentration. The reason for this effect is unknown but has been previously observed in these molecules by others [52] . It is speculated that the addition of both the peptide and PEG linker affects the staining of the DNA.

[0042] FIGURE 27B depicts a denaturing PAGE gel showing time-dependent 3CL- mediated cleavage of FQ-3CL-CLIP. Lane 1 shows the intact FQ-3CL-CLIP, while lanes 2-7 represent samples incubated with 1 pM 3CL protease for vary ing durations: 15 min, 30 min, 1 h, 2 h, 4 h. and 6 h, respectively. Approximately 2 pmol of each sample was loaded onto the gel, which was imaged using the Cy5 fluorescence channel. Following protease treatment, a new band with higher mobility' appears, corresponding to the cleaved fragment. Over time, the intensity⁷ of the original FQ-3CL-CLIP band diminishes, while the intensity' of the cleaved fragment band increases, indicating progressive and successful proteolytic cleavage of FQ- 3CL-CLIP. It should be noted that the intact CLIP structure contains both Cy5 and its quencher. BHQ. As the CLIP degrades, Cy5 and BHQ separate, causing an increase in Cy5 fluorescence relative to the intact structure. This artificially enhances the intensity' of the fragment bands in the Cy5 channel, making the proportion of fragments appear higher than it actually is.

[0043] FIGURE 27C depict a denaturing PAGE gel showing selectivity’ of FQ-3CL-CLIP. Lanes 1 and 4 show untreated FQ-3CL-CLIP samples after 2 h and 4 h, respectively. Lanes 2 and 5 represent FQ-3CL-CLIP incubated with 1 pM 3CL protease for 2 h and 4 h, respectively. Lanes 3 and 6 correspond to FQ-3CL-CLIP incubated with 1 pM CTSB protease for 2 h and 4 h, respectively. Approximately 2 pmol of each sample was loaded onto the gel. After incubation with 3CL protease, anew band with higher mobility appears, indicating successful cleavage of FQ-3CL-CLIP. Over time, the intensity of the original FQ-3CL-CLIP band diminishes, while the intensity' of the cleaved fragment band increases, indicating progressive and successful proteolytic cleavage of FQ-3CL-CLIP. In contrast, incubation with CTSB protease also generates a new band, but with much lower intensity relative to the 3CL protease-treated samples. This observation highlights the preference of 3CL protease for FQ-3CL-CLIP over CTSB protease, demonstrating the higher selectivity' of 3CL protease for this substrate. It should be noted that the intact CLIP structure contains both Cy5 and its quencher, BHQ. As the CLIP degrades, Cy5 and BHQ separate, causing an increase in Cy5 fluorescence relative to the intact structure. This artificially enhances the intensity of the fragment bands in the Cy5 channel, making the proportion of fragments appear higher than it actually is.

[0044] FIGURE 28 depicts fluorescence enhancement observed from treatment of 10 nM RNP with varied concentrations of the Initiator DNA after 30 min. Fluorescence enhancement refers to the fluorescence of a sample relative to the fluorescence of the RNP. *, **, and *** denote statistical significance at the 95%, 99%, and 99.9% confidence levels, respectively, assessed using a one-tailed Student’s t-test.

[0045] FIGURE 29 depicts fluorescence enhancement observed after 5 min when CRISPR RNP and DNase Alert reporters are treated with Initiator DNA, DNA Hairpin 10-8, DNA Hairpin 30-8, and CLIP, respectively. Fluorescence enhancement is calculated as the fluorescence observed relative to the fluorescence of the RNP alone, which is set to a value of 1. *, and *** denote statistical significance at the 95%, and 99.9% confidence levels, respectively, assessed using a one-tailed Student’s t-test.

[0046] FIGURES 30A-30C depict a hypothesized mechanism of activation of CRISPR- Casl2a RNP via DNA hairpins treated with Blocking DNA. FIG. 30A shows that introducing 24-nt Blocking DNA, which is fully complementary to the hairpin's Initiator DNA sequence, effectively inactivates CRISPR. This inactivation occurs because the 24-nt Blocking DNA opens the hairpin through toehold-mediated strand displacement. FIG. 30B shows that adding 16-nt Blocking DNA, which covers only part of the Initiator DNA sequence and not the hairpin stem, initially diminishes the CRISPR signal. Over time, however, the signal increases, resembling a positive cooperativity curve. This observation could be attributed to factors such as DNA that is misfolded and not in hairpin form or slow disruption of the hairpin stem by CRISPR-Casl2a due to strand displacement. FIG. 30C shows that, with 16-nt Blocking DNA, once initial activation occurs, the CRISPR-Casl2a enzymes, capable of cleaving ssDNA, begin to degrade the hairpin's loop region. This degradation releases new Initiator DNA, leading to an amplification cycle.

[0047] FIGURE 31 depicts the UV-Vis spectra of FQ-3CL-CLIP after gel purification. Characteristic absorbances of DNA, BHQ, and cyanine-5 at 260 nm, -580 nm, -650 nm respectively, are observed while the absence of DBCO's characteristic 310 nm peak indicates successful conjugation of quencher-DNA linker species and dye labeled blocking strands.

[0048] FIGURE 32 depicts the scheme of activation of FQ-3CL-CLIPs due to proteolytic cleavage.

[0049] FIGURE 33 depicts a comparison of fluorescence signal obtained from 3CL-CLIP assays and 3CL-FQ-CLIP. Relative fluorescence refers to the ratio of the fluorescence of a sample to the fluorescence of the probe without the 3CL protease. The fluorescence of the probe without 3CL protease set to a value of 1. The final probe concentration in the assay is indicated in the figure. *, **, and *** denote statistical significance at the 95%, 99%, and 99.9% confidence levels, respectively, assessed using a one-tailed Student's t-test.

[0050] FIGURE 34A depicts a comparison of fluorescence enhancement when CTSB- CLIP and CTSB-CFP are treated with 10 nM CTSB. Fluorescence enhancement refers to ratio of the fluorescence of a sample to the fluorescence of the RNP alone, which is set to a value of 1. The final probe concentration in the assay is indicated in the figure. FIGURE 34B depicts the fluorescence response of CTSB-CFP upon incubation with CTSB protease for 30 min. *, **, and *** denote statistical significance at the 95%, 99%, and 99.9% confidence levels, respectively, while "ns" indicates no statistical significance (at the 95% confidence level), assessed using a one-tailed Student's t-test.

[0051] FIGURE 35 depicts fluorescence enhancement resulting from FQ-3CL-CLIP being treated with DNase I. Fluorescence enhancement refers to ratio of the fluorescence of a sample to the fluorescence of the FQ-3CL-CLIP alone, which is set to a value of 1.

[0052] FIGURE 36 depicts a denaturing PAGE gel of FQ-3CL-CLIPs treated with cell lysates. The gel was imaged in the Cy5 channel, stained with GelRed, and reimaged in the GelRed channel. Bands in the Cy5 channel result from the fluorophore in the FQ-3CL-CLIP whereas bands in the GelRed channel result from the binding of GelRed to the DNA sequences. Panels (1-4) show samples treated with cellular lysates for 30 min, 1 h, 3 h, and 6 h, respectively, while panel (5) shows FQ-3CL-CLIP without cellular lysate treatment. Approximately 2 pmol of each sample was loaded.

[0053] FIGURE 37A shows detection of 1 pM 3CL protease spiked into NCI-H508 cell lysates using 3CL-CLIP. FIGURE 37B shows activation of CTSB-CLIPs when treated with 15 pL of lysates from RKO cell line. 3CL-CLIP is used as a control. Fluorescence enhancement is defined as ratio of the fluorescence values of a sample to the fluorescence of the RNP complex. FIGURE 37C shows signal generated when 100 nM CTSB-CFP is treated with 6 pL of RKO cell lysates in the presence and absence of a CTSB inhibitor. FIGURE 37D shows signal generated when 1 nM CTSB-CLIP is treated with 6 pL of RKO cell lysates in the presence and absence of a CTSB inhibitor. *, **, and *** denote statistical significance at the 95%, 99%, and 99.9% confidence levels, respectively, while "ns" indicates no statistical significance (at the 95% confidence level), assessed using a one-tailed Student's t-test.

[0054] FIGURE 38 depicts a comparison of fluorescence enhancement from 3CL-CLIP and 3CL-CFP treated with 1 pM 3CL protease, with and without 10% serum. Relative fluorescence is calculated as the ratio of the fluorescence of each sample to that of the probe without protease after 15 min, with the fluorescence of probes without protease set to 1. **, and *** denote statistical significance at the 99%, and 99.9% confidence levels, respectively using a one-tailed Student's t-test.

[0055] FIGURES 39A-39B depict the response of CTSB-CLIP to varying amounts of SW-620 colon cancer cell lysates. 10 nM of CTSB-CLIP was incubated with 0 pL, 3 pL (FIG. 39 A), or 15 pL (FIG. 39B) of the cell lysates (~2 million cells/mL) in a total volume of 45 pL of IX PBS. The samples were then incubated for 30 min with shaking at RT. Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Cast 2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final CTSB-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read over time (excitation wavelength of 525 nm and an emission at 566 nm). It was observed that volumes below 3 pL of cell lysates did not return appreciable responses and therefore the experimental LOD is approximately 6,000 cells.

[0056] FIGURE 40 depicts fluorescence enhancement of CTSB-CFP after incubation with lysates of HT29. NCI-H508. RKO, and SW-620 cell lines. 1 pM of CTSB-CFP was incubated with 15 pL of various cell lysates (~2 million cells/mL) in a total volume of 45 pL of IX PBS. The samples were then incubated for 30 min with shaking at RT. Following this, the samples were diluted 10-fold with CRISPR Buffer. Therefore, the final CTSB-CFP concentration at fluorescence reading was 100 nM. The fluorescence from CTSB-CFP read after an additional 30 min (excitation wavelength of 380 nm and an emission at 460 nm). Fluorescence enhancement refers to the ratio of the fluorescence of a sample to the fluorescence of the buffer, which is set at a value of 1. *** denotes statistical significance at the 99.9% confidence level while "ns" indicates no statistical significance (at the 95% confidence level), assessed using a one-tailed Student's t-test.

DETAILED DESCRIPTION

[0057] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

DEFINITIONS

[0058] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0059] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,’' “included;’ “involving,"’ “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0060] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0061] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0062] When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. "about x, y, z, or less’ and should be interpreted to include the specific ranges of ’about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z' as well as the ranges of ‘greater than x', greater than y', and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”. where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ’y’”.

[0063] It is to be understood that such a range format is used for convenience and brevity⁷, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0064] As used herein, the terms “about,” “approximate.” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off. measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0065] Reference is made herein to nucleic acid and nucleic acid sequences. The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be singlestranded or double-stranded and may represent the sense or the antisense strand). This includes modified nucleic acids. The nucleic acid sequence can include any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil (U), adenine (A), thymine (T), cytosine (C), guanine (G), inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc. It may include modified bases, including locked nucleic acids, peptide nucleic acids and others known to those skilled in the art.

[0066] An "oligonucleotide" is a polymer including two or more nucleotides. The polymer can additionally include non-nucleotide elements such as labels, quenchers, blocking groups, or the like. The nucleotides of the oligonucleotide can be natural or non-natural and can be unsubstituted, unmodified, substituted or modified. The nucleotides can be linked by phosphodiester bonds, or by phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, or the like.

[0067] Reference also is made herein to peptides, polypeptides, proteins and compositions including peptides, polypeptides, and proteins. As used herein, a polypeptide and/or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).

[0068] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and/or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereol).

[0069] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S). threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, 0-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid. 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutync acid. 4-Hydroxyproline, pipendimc acid. 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine, 3 -Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid. 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine. Norvaline, 2,2'-Diaminopimelic acid. Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0070] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alky lation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g.. the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g.. the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenz matic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g.. of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, ty rosine, threonine or histidine).

[0071] Variants including deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5 '-terminal or 3 '-terminal truncation or both of a reference polynucleotide).

[0072] Variants including a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may include up to the entire length of the reference sequence, minus at least one nucleotide/amino acid residue. For example, a fragment may include from 4 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may include at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and/or the C-terminal region of a polypeptide or the 5'-terminal region and/or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full-length polynucleotide or full length polypeptide.

[0073] Variants including insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150. or 200 amino acid residues or nucleotides.

[0074] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N- terminus. the C-terminus, or both termini. A fusion protein includes at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0075] A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3' end of a first polynucleotide to a 5’ end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below). [0076] “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences or polynucleotide sequences. Homology, sequence similarity’, and percentage sequence identity may be determined using methods in the art and described herein.

[0077] The phrases “percent identity ” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda. Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

[0078] Percent identity may be measured over the length of an entire defined polypeptide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 1 , at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0079] A “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%. at least 91%. at least 92%. at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide.

[0080] A variant polypeptide may have substantially the same functional activity as a reference polypeptide. For example, a variant polypeptide may exhibit or more biological activities associated with binding a ligand and/or binding DNA at a specific binding site.

[0081] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety ). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0082] Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplar}' only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0083] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0084] A “variant,” “mutant.” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the ‘"BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), ‘‘Blast 2 sequences — a new⁷ tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show; for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%. or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0085] Nucleic acid sequences that do not show⁷ a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0086] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0087] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. [0088] “Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods w ell knowm in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukary otic host cell. The method for transformation is selected based on the type of host cell being transformed and may⁷ include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells’" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0089] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified"’ refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they⁷ are naturally associated.

[0090] The term “mismatched” or “mismatched target sequence” refers to an off-target sequence that is not perfectly complementary to the first DNA sequence or the second DNA sequence of the chimeric deoxyribonucleic acid described herein. The dual retargeted DNA may have at least one mismatch, but can also have 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides to the off-target sequence.

[0091] As used herein, the term “molecular beacon” refers a to detectable molecule, where the detectable property of the molecule is detectable only under certain specific conditions, thereby enabling it to function as a specific and informative signal. Non-limiting examples of detectable properties are optical properties, electrical properties, magnetic properties, chemical properties and time or speed through an opening of known size. In some embodiments a molecular beacon can be a single-stranded oligonucleotide capable of forming a stem-loop structure, where the loop sequence may be complementary to a target nucleic acid sequence of interest and is flanked by short complementary' arms that can form a stem. The oligonucleotide may be labeled at one end with a fluorophore and at the other end with a quencher molecule. In the stem-loop conformation, energy from the excited fluorophore is transferred to the quencher, through long-range dipole-dipole coupling similar to that seen in fluorescence resonance energy transfer, or FRET, and released as heat instead of light. When the loop sequence is hybridized to a specific target sequence, the two ends of the molecule are separated and the energy from the excited fluorophore is emitted as light, generating a detectable signal. [0092] As used herein, the term “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term “pharmaceutically acceptable” is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0093] As used herein, the term “detecting” used in context of detecting a signal from a detectable label to indicate the presence of a target nucleic acid (such as a signaling nucleic acid) in the sample does not require the method to provide 100% sensitivity and/or 100% specificity. As is well known, “sensitivity” is the probability that a test is positive, given that the sample has a target nucleic acid sequence, while “specificity” is the probability that a test is negative, given that the sample does not have the target nucleic acid sequence. A sensitivity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. A specificity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. Detecting also encompasses assays with false positives and false negatives. False negative rates may be 1%, 5%, 10%, 15%, 20% or even higher. False positive rates may be 1%, 5%, 10%, 15%, 20% or even higher. The term “detecting” is also used in the context of detecting the amplified target nucleic acid by its melting temperature using melting curve analysis, as is known in the art.

[0094] As used herein, “labels” are chemical or biochemical moieties useful for labeling a nucleic acid (including a single nucleotide), amino acid, or antibody. “Labels” include fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionuclides, enzymes, substrates, cofactors, inhibitors, magnetic particles, quantum dots, and other moieties known in the art. “Labels” are capable of generating a measurable signal, or can be used to capture nucleic acids, and may be covalently or noncovalently joined to an oligonucleotide or nucleotide (e.g., a non-natural nucleotide).

COMPOSITIONS

[0095] Disclosed herein is a new class of chemical probes, termed DNA-locked beacons, for enzyme (e.g., protease) activity detection with, for example, CRISPR-Casl2a-amplified signaling. Upon exposure to specific enzymes, the cleavage of a variable cleavable region causes the DNA-locked beacon to undergo a conformational change, transitioning from a double-stranded hairpin structure to single-stranded DNA. This change results in differences in the steric hindrance of the probes such that when cleaved, they can access and activate CRISPR-Casl2a enzymes (for example) without impediment, and, when paired with a DNase reporter, enable robust signal detection and amplification. Using the 3CL Main protease of SARS-CoV-2 as an example, it was demonstrated that 3CL-specific DNA-locked peptide beacons resulted in 70-fold signal turn on, significantly outperforming commercial fluorophore quencher probes which show no signal increase at the same concentration. Moreover, the versatility of DNA-locked beacons is showcased by effectively detecting multiple diseaserelevant proteases, including caspase-3, MMP7, and cathepsin B, within a rapid assay time of 35-45 minutes.

[0096] Also disclosed is the ability of these probes (also referred to herein as “DNA-locked beacons” or “DNA-locked peptide beacons” or “DLPB” or just “beacons”, as well as “DNA hairpin sensors” or just “DNA Hairpins” or “sensors”) to work in patient-derived cancer cell lysates, where they demonstrate the ability to detect active enzy mes (e.g., proteases) in complex media. Importantly, the DNA hairpin sensors overcome existing challenges in detecting enzyme activity. They eliminate the need for nanoparticle attachment and specialized equipment, offering a straightforward, amplifiable method. These sensors enable efficient enzyme detection at 25°C, for example, using a simple fluorescence readout, streamlining the process significantly. It is noted that they can be designed to work in a variety of conditions that can be varied, such as pH, temperature, etc. The unique structural transition of these sensors makes them ideal for advanced enzymatic detection and are adaptable to other CRISPR-Casl2a based detection systems. DNA-locked beacons offer an innovative method to detect enzyme activity where conventional detection techniques are suboptimal.

[0097] Disclosed herein are DNA-locked beacons and methods of using them. (It is noted that while it is referred to as a DLPB, the DNA can actually be any nucleic acid, and the cleavable domain can be any cleavable molecule besides a peptide) Specifically, the beacons of the present invention include the following components: (1) a cleavable domain (e.g., a peptide domain) that is recognized and cleaved by the enzyme of interest, (2) a “signaling” nucleic acid sequence (also referred to herein as an initiator sequence) that can produce a detectable signal, for example, by⁷ activating a signaling event such as the Casl2a or Casl3a RNP system or amplification of a barcode nucleic acid sequence, and (3) a short “blocking” nucleic acid. Optionally, the beacon can also include a linker sequence (4). Examples of these specific components and their sequences can be found in the following TABLES 1-3. Contemplated herein are the beacons themselves, the components making up the beacons, and compositions which include variation to these beacons and their components, as described in the definitions section above, as well as below.

[0098] The beacons described herein include a combination of peptide sequence and nucleic acid sequence. Also disclosed herein are variants of these peptide/nucleic acid sequence. For example, a beacon can comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%. 90%. 95%, 96%, 97%, 98%, or 99% sequence identity to any nucleic acid/amino acid sequence disclosed herein. Variant nucleic acids, peptides, polypeptides, and proteins may include nucleic acid, peptides, polypeptides, and proteins having one or more substitutions, deletions, additions and/or amino acid/nucleic acid insertions relative to a reference nucleic acid, peptide, polypeptide, or protein. Examples of these sequences are provided below.

[0099] In an aspect, provided is a nucleic acid-locked beacon, wherein said nucleic acid- locked beacon includes: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary' to the signaling nucleic acid sequence. [0100] In another aspect, provided is a composition including a nucleic acid-locked beacon, wherein said nucleic acid-locked beacon includes: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence.

[0101] In yet another aspect, provided is a composition including two or more nucleic acid- locked beacons, wherein each of said nucleic acid-locked beacon includes: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary' to the signaling nucleic acid sequence; wherein the two or more nucleic acid- locked beacons include different peptide sequences. In some aspects, the two or more nucleic acid-locked beacons can allow for the detection of two or more different enzymes in the same assay (multiplex).

[0102] In yet another aspect, provided is a diagnostic assay for determining presence of an enzyme, the assay including a nucleic acid-locked beacon, wherein said nucleic acid-locked beacon includes: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary' to the signaling nucleic acid sequence. In some aspects, the diagnostic assay can include two or more nucleic acid-locked beacons, and the tyvo or more nucleic acid-locked beacons can include different peptide sequences. In some such aspects, the tyvo or more nucleic acid-locked beacons can allow for the detection of two or more different enzymes in the same assay (multiplex). When used as a multiplex, 2. 3. 4, 5, 6. 7, or more different enzymes can be detected at the same time. These enzymes can be distinguished from each other by different signaling nucleic acids.

[0103] In some aspects, the cleavable domain can be recognized and can be cleavable by an enzyme of interest. In some aspects, the enzyme of interest can be a protease of interest. In some such aspects, the protease of interest can be MMP7, Caspase 3, 3CL, or Cathepsin B, although the invention is useful with any protease that can be detected upon cleavage of its target peptide, which can be used as the “baif ’ in the peptide domain. A comprehensive list of proteases can be found in Rawlings, N.D., Barrett, A.J., Thomas, P.D., Huang, X., Bateman, A. & Finn, R.D. (2018) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46, D624-D632. These proteases are herein incorporated by reference in their entirety. In other aspects, the enzyme of interest can be another hydrolase (e.g., esterase, nuclease, phosphodiesterase, lipase, phosphatase, glycosylase, glycoside hydrolase, helicase, GTPase, lipase, glycosidase, peptidase, nucleosidase, or any other hydrolase which could be identified by one of ordinary skill in the art).

[0104] In some aspects, the cleavable domain can be selected based on the enzyme of interest. For example, if the enzyme of interest is a protease, the cleavable domain can be a peptide domain. As another example, if the enzyme of interest is a glycosidase, the cleavable domain can be a sugar domain. Appropriate classes of cleavable domains for any given enzyme of interest could be substantially identified by one of ordinary skill in the art.

[0105] In some specific aspects, the cleavable domain can be a peptide domain. In some aspects, the peptide domain can be at least 3 amino acids (e g., at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids) in length. In some aspects, the peptide domain can be up to 75 amino acids (e.g., up to 70 amino acids, up to 65 amino acids, up to 60 amino acids, up to 55 amino acids, up to 50 amino acids, up to 45 amino acids, up to 40 amino acids, up to 35 amino acids, up to 30 amino acids, up to 25 amino acids, up to 20 amino acids, up to 15 amino acids, up to 15 amino acids, up to 10 amino acids, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids) in length. In some aspects, the peptide domain can be more than 75 amino acids in length.

[0106] It is considered that the peptide domain can be any number of amino acids in length ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the peptide domain can be from 3 to 75 amino acids (e.g., from 4 to 70 amino acids, from 5 to 65 amino acids, from 6 to 60 amino acids, from 7 to 55 amino acids, from 8 to 50 amino acids, from 9 to 45 amino acids, from 10 to 40 amino acids, from 15 to 35 amino acids, from 20 to 30 amino acids, from 3 to 25 amino acids, from 4 to 20 amino acids, from 5 to 15 amino acids, from 6 to 10 amino acids, from 7 to 9 amino acids, from 25 to 75 amino acids, from 30 to 70 amino acids, from 35 to 65 amino acids, from 40 to 60 amino acids, from 45 to 55 amino acids) in length.

[0107] In some aspects, the peptide domain can include 80% similarity or more (e.g., 81% similarity or more. 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more. 86% similarity or more. 87% similarity or more. 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to any one of SEQ ID NOs: 1-4. In some aspects, the peptide domain can include any one of SEQ ID NOs: 1-4.

[0108] In some aspects, the signaling nucleic acid sequence can be configured to produce a detectable signal. In some such aspects, the detectable signal can be a fluorescence signal. For example, in some aspects, the signaling nucleic acid sequence can activate a Cas molecule, and the Cas molecule can cleave a nucleic acid reporter, thereby causing the fluorescence signal. In some such aspects, the Cas molecule can include a Casl2a or Casl3a RNP system. In this example, the signaling nucleic acid sequence can activate a Cas-guide RNA complex (also called a “ribonucleoprotein” or “RNP”), and the RNP can cleave the nucleic acid reporter, thereby causing the fluorescence signal. For example, the signaling nucleic acid sequence can hybridize to the guide RNA complexed to an inactive Cas molecule. This can activate the Cas molecule and allow it to cleave a reporter. In some such aspects, the reporter can include a fluorophore and a quencher, wherein the quencher can prevent detection of the fluorophore when the reporter is uncleaved, and wherein the fluorophore can be detected when the reporter is cleaved by the activated Cas molecule. In some aspects, the reporter can be a DNase or RNase reporter. In some aspects, the reporter can be any DNase or RNase reporter that provides a signal upon nucleic acid cleavage.

[0109] Additionally or alternatively, in other such aspects, the signaling nucleic acid sequence can further include a barcode nucleic acid sequence and/or a primer site for amplif ing said barcode nucleic acid sequence, and the detectable signal can be said amplified barcode nucleic acid sequence. For example, in some aspects, the barcode nucleic acid sequence can be amplified by any suitable nucleic acid amplification platform, for example, polymerase chain reaction (PCR), exponential amplification reaction (EXPAR), loop-mediated isothermal amplification (LAMP), next-generation sequencing (NGS), strand displacement amplification (SDA), or hybridization chain reaction.

[0110] In some aspects, the signaling nucleic acid sequence can be about 4 or more nucleotides in length (e.g., at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides) in length. In some aspects, the signaling nucleic acid sequence can be up to 100 nucleotides (e.g., up to 95 nucleotides, up to 90 nucleotides, up to 85 nucleotides, up to 80 nucleotides, up to 75 nucleotides, up to 70 nucleotides, up to 65 nucleotides, up to 60 nucleotides, up to 55 nucleotides, up to 50 nucleotides, up to 45 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides, up to 25 nucleotides, up to 20 nucleotides, up to 15 nucleotides, up to 10 nucleotides, up to 9 nucleotides, up to 8 nucleotides, up to 7 nucleotides, up to 6 nucleotides, up to 5 nucleotides, up to 4 nucleotides) in length. In some aspects, the signaling nucleic acid sequence can be longer than 100 nucleotides in length. [OHl] It is considered that the signaling nucleic acid sequence can be any length ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the signaling nucleic acid sequence can be from 4 to 100 nucleotides (e.g., from 5 nucleotides to 95 nucleotides, from 6 nucleotides to 90 nucleotides, from 7 nucleotides to 85 nucleotides, from 8 nucleotides to 80 nucleotides, from 9 nucleotides to 75 nucleotides, from 10 nucleotides to 70 nucleotides, from 15 nucleotides to 65 nucleotides, from 20 nucleotides to 60 nucleotides, from 25 nucleotides to 55 nucleotides, from 30 nucleotides to 50 nucleotides, from 35 nucleotides to 45 nucleotides, from 4 nucleotides to 40 nucleotides, from 5 nucleotides to 35 nucleotides, from 6 nucleotides to 30 nucleotides, from 7 nucleotides to 25 nucleotides, from 8 nucleotides to 20 nucleotides, from 9 nucleotides to 15 nucleotides, from 40 nucleotides to 100 nucleotides, from 45 nucleotides to 95 nucleotides, from 50 nucleotides to 90 nucleotides, from 55 nucleotides to 85 nucleotides, from 60 nucleotides to 80 nucleotides, from 65 nucleotides to 75 nucleotides).

[0112] In some aspects, the signaling nucleic acid sequence can include DNA. Additionally or alternatively, in other aspects, the signaling nucleic acid can include RNA. The signaling nucleic can also include non-naturally occurring nucleic acids (such as nucleic acids with base, backbone, or sugar modifications), non-nucleic acids, or tags, reporters, barcodes, or other methods that can be used for its detection.

[0113] In some aspects, the signaling nucleic acid sequence can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more. 90% similarity or more. 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity’ or more. 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity’ or more) to any one of SEQ ID NOs: 6, 8, or 10. In some aspects, the signaling nucleic acid sequence can include any one of SEQ ID NOs: 6, 8, or 10.

[0114] The function of the blocking nucleic acid is to hybridize with the signaling nucleic acid, and therefore prevent the signaling nucleic acid from creating a detectable signal, until the enzyme has interacted (cleaved) the cleavable domain of the beacon. In some aspects, the blocking nucleic acid can be at least 4 nucleotides (e.g., at least 6 nucleotides, at least 8 nucleotides, at least 10 nucleotides, at least 12 nucleotides, at least 14 nucleotides, at least 16 nucleotides, at least 18 nucleotides, at least 20 nucleotides) in length. In some aspects, the blocking nucleic acid can be up to 20 nucleotides (e g., up to 18 nucleotides, up to 16 nucleotides, up to 14 nucleotides, up to 12 nucleotides, up to 10 nucleotides, up to 8 nucleotides, up to 6 nucleotides, up to 4 nucleotides) in length. In some aspects, the blocking nucleic acid can be more than 20 nucleotides in length. It is contemplated that the blocking nucleic acid can be any length which is appropriate for hybridization (and blocking) of the signal nucleic acid.

[0115] It is considered that the blocking nucleic acid can be any length ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the blocking nucleic acid can be from 4 to 20 nucleotides (e.g., from 6 to 18 nucleotides, from 8 to 16 nucleotides, from 10 to 14 nucleotides, from 4 to 12 nucleotides, from 6 to 10 nucleotides, from 12 to 20 nucleotides, from 14 to 18 nucleotides) in length.

[0116] In some aspects, the blocking nucleic acid sequence can include DNA. Additionally or alternatively, in other aspects, the blocking nucleic acid can include RNA. It can also include non-naturally occurring nucleic acids (such as nucleic acids with base, backbone, or sugar modifications) or other elements which allow it to block the signaling nucleic acid.

[0117] In some aspects, the blocking nucleic acid sequence can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more. 89% similarity or more. 90% similarity or more. 91% similarity’ or more, 92% similarity⁷ or more, 93% similarity or more, 94% similarity' or more, 95% similarity or more, 96% similarity or more, 97% similarity’ or more, 98% similarity or more, 99% similarity or more) to any one of SEQ ID NOs: 7, 9, or 14-15. In some aspects, the blocking nucleic acid sequence can include any one of SEQ ID NOs: 7, 9, or 14-15.

[0118] In some aspects, the signaling nucleic acid sequence can be adjacent (i.e., either immediately adjacent or separated by a linker) a 5’ end of the cleavable domain, and the blocking nucleic acid sequence can adjacent a 3‘ end of the cleavable domain. In other aspects, the signaling nucleic acid sequence can be adjacent a 3’ end of the cleavable domain, and the blocking nucleic acid sequence can be adjacent a 5’ end of the cleavable domain.

[0119] In some aspects, when the cleavable domain is intact, the nucleic acid portion of the beacon can form a secondary’ structure selected from a hairpin structure and a circularized structure.

[0120] In some aspects, when the cleavable domain is intact, the blocking nucleic acid sequence and the signaling nucleic acid sequence can be at least partially hybridized. By “partially hybridized” means that at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides are hybridized between the blocking nucleic acid and the signaling nucleic acid sequence. In some such aspects, when the cleavable domain is intact, the blocking nucleic acid sequence and the signaling nucleic acid sequence can be at least partially hybridized under a given set of conditions, and further, upon cleavage of the cleavable domain by the enzyme, the blocking nucleic acid sequence and the signaling sequence can at least partially denature under said given set of conditions. This denaturation can allow for detection of the signaling nucleic acid sequence.

[0121] In some aspects, the given set of conditions can include temperature, pH, and/or salt concentration. For example, in some aspects, the temperature of the given conditions can be at least about 4°C (e.g., at least about 5°C, at least about 10°C, at least about 15°C, at least about 20°C, at least about 25°C, at least about 30°C. at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C. at least about 55°C, at least about 60°C, at least about 65°C. at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C). In some aspects, the temperature of the given conditions can be up to about 90°C (e.g., up to about 85°C, up to about 80°C, up to about 75°C, up to about 70°C, up to about 65°C, up to about 60°C, up to about 55°C, up to about 50°C, up to about 45°C, up to about 40°C, up to about 35°C, up to about 30°C, up to about 25°C, up to about 20°C, up to about 15°C, up to about 10°C, up to about 5°C, up to about 4°C).

[0122] It is considered that the temperature of the given conditions can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the temperature of the given conditions can be from about 4°C to about 90°C (e.g., from about 5°C to about 85°C, from about 10°C to about 80°C, from about 15°C to about 75°C. from about 20°C to about 70°C, from about 25°C to about 65°C. from about 30°C to about 60°C, from about 35°C to about 55°C, from about 40°C to about 50°C, from about 4°C to about 45°C, from about 5°C to about 40°C, from about 10°C to about 35°C, from about 15°C to about 30°C, from about 20°C to about 25°C, from about 45°C to about 90°C, from about 50°C to about 85°C, from about 55°C to about 80°C. from about 60°C to about 75°C, from about 65°C to about 70°C).

[0123] In some aspects, the pH of the given conditions can be at least about 4 (e.g., at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.5, at least about 10). In some aspects, the pH of the given conditions can be up to about 10 (e.g., up to about 9.5, up to about 9, up to about 8.5, up to about 8, up to about 7.5, up to about 7, up to about 6.5, up to about 6, up to about 5.5, up to about 5, up to about 4.5, up to about 4).

[0124] It is considered that the pH of the given conditions can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the pH of the given conditions can be from about 4 to about 10 (e.g., from about 4.5 to about 9.5, from about 5 to about 9, from about 5.5 to about 8.5, from about 6 to about 8, from about 6.5 to about 7.5, from about 4 to about 7, from about 4.5 to about 6.5, from about 5 to about 6, from about 7 to about 10, from about 7.5 to about 9.5, from about 8 to about 9).

[0125] In some aspects, the salt concentration of the given conditions can be at least about 2 mM (e.g., at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 150 mm, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM). In some aspects, the salt concentration of the given conditions can be up to about 500 mM (e.g., up to about 450 mM, up to about 400 mM, up to about 350 mM, up to about 300 mM, up to about 250 mM, up to about 200 mM, up to about 150 mM, up to about 100 mM, up to about 90 m , up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM. up to about 40 mM, up to about 30 mM, up to about 20 mM. up to about 10 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM).

[0126] It is considered that the salt concentration of the given conditions can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the salt concentration of the given conditions can be from about 2 mM to about 500 mM (e.g., from about 3 mM to about 450 rnM, from about 4 mM to about 400 mM, from about 5 mM to about 350 mM, from about 10 mM to about 300 mM, from about 20 mM to about 250 mM, from about 30 mM to about 200 mM, from about 40 mM to about 150 mM, from about 50 mM to about 100 mM, from about 60 mM to about 90 mM, from about 70 rnM to about 80 mM, from about 2 mM to about 80 rnM, from about 3 mM to about 70 mM, from about 4 rnM to about 60 mM, from about 5 mM to about 50 mM, from about 10 mM to about 40 mM, from about 20 mM to about 30 mM, from about 70 mM to about 500 rnM, from about 80 mM to about 450 mM, from about 90 mM to about 400 mM, from about 100 mM to about 350 mM. from about 150 mM to about 300 mM, from about 200 mM to about 250 mM). [0127] In some aspects, at least partial denaturation of the blocking nucleic acid sequence and signaling nucleic acid sequence can allow the signaling nucleic acid sequence to produce a detectable signal. In some aspects, at least partial hybridization of the blocking nucleic acid sequence and the signaling nucleic acid sequence can at least partially reduce production of a detectable signal by the signaling nucleic acid sequence. By way of specific example, before cleavage of the cleavable domain by an enzyme, the nucleic acid-locked beacon can have a melting temperature of above 25°C, resulting in the signaling nucleic acid sequence and the blocking nucleic acid sequence at least partially hybridizing at room temperature. When at least partially hybridized, the signaling nucleic acid sequence may not be able to produce a detectable signal. For example, the blocking nucleic acid sequence may block the signaling nucleic acid sequence from interacting with a Cas system or an amplification system (such as PCR) to produce a detectable signal. After cleavage of the cleavable domain by an enzyme, the resulting double-stranded complex can have a melting temperature of less than 25°C, resulting in the signaling nucleic acid sequence and the blocking nucleic acid sequence at least partially separating into two individual strands at room temperature. The separating of the two strands can allow the signaling nucleic acid to produce a detectable signal, for example, via a Cas system or a nucleic acid amplification system as discussed in detail below.

[0128] It is considered that, in some aspects, additionally or alternatively, the blocking nucleic acid sequence may produce a detectable signal. [0129] In some aspects, the nucleic acid-locked beacon can further include a linker. In some such aspects, the linker can be between the cleavable domain and the blocking nucleic acid. In other such aspects, the linker can be between the cleavable domain and the signaling nucleic acid sequence.

[0130] In some aspects, the linker can include one or more bifunctional molecules. For example, in some aspects, the linker can include amino acids, nucleic acids (e.g., DNA or RNA), polymers (e.g.. PEG), or any combination thereof. In some such aspects, a portion or all of the linker can include a peptide portion. For example, in some aspects, the peptide portion of the linker can be at least 3 amino acids (e.g., at least 4 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 95 amino acids, at least 100 amino acids) in length. In some aspects, the peptide portion of the linker can be up to 100 amino acids (e.g., up to 95 amino acids, up to 90 amino acids, up to 85 amino acids, up to 80 amino acids, up to 75 amino acids, up to 70 amino acids, up to 65 amino acids, up to 60 amino acids, up to 55 amino acids, up to 50 amino acids, up to 45 amino acids, up to 40 amino acids, up to 35 amino acids, up to 30 amino acids, up to 25 amino acids, up to 20 amino acids, up to 15 amino acids, up to 10 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids) in length.

[0131] It is considered that the peptide portion of the linker can have a length ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the peptide portion of the linker can be from 3 to 100 amino acids (e.g., from 4 to 95 amino acids, from 5 to 90 amino acids, from 10 to 85 amino acids, from 15 to 80 amino acids, from 20 to 75 amino acids, from 25 to 70 amino acids, from 30 to 65 amino acids, from 35 to 60 amino acids, from 40 to 55 amino acids, from 45 to 50 amino acids, from 3 to 50 amino acids, from 4 to 45 amino acids, from 5 to 40 amino acids, from 10 to 35 amino acids, from 15 to 30 amino acids, from 20 to 25 amino acids, from 45 to 100 amino acids, from 50 to 95 amino acids, from 55 to 90 amino acids, from 60 to 85 amino acids, from 65 to 80 amino acids, from 70 to 75 amino acids) in length.

[0132] In other such aspects, a portion or all of the linker can include a nucleic acid portion. For example, in some aspects, the nucleic acid portion of the linker can be at least 3 nucleotides (e.g., at least 4 nucleotides, at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides) in length. In some aspects, the nucleic acid portion of the linker can be up to 100 nucleotides (e.g., up to 95 nucleotides, up to 90 nucleotides, up to 85 nucleotides, up to 80 nucleotides, up to 75 nucleotides, up to 70 nucleotides, up to 65 nucleotides, up to 60 nucleotides, up to 55 nucleotides, up to 50 nucleotides, up to 45 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides, up to 25 nucleotides, up to 20 nucleotides, up to 15 nucleotides, up to 10 nucleotides, up to 5 nucleotides, up to 4 nucleotides, up to 3 nucleotides) in length.

[0133] It is considered that the nucleic acid portion of the linker can have a length ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nucleic acid portion of the linker can be from 3 to 100 nucleotides (e.g., from 4 to 95 nucleotides, from 5 to 90 nucleotides, from 10 to 85 nucleotides, from 15 to 80 nucleotides, from 20 to 75 nucleotides, from 25 to 70 nucleotides, from 30 to 65 nucleotides, from 35 to 60 nucleotides, from 40 to 55 nucleotides, from 45 to 50 nucleotides, from 3 to 50 nucleotides, from 4 to 45 nucleotides, from 5 to 40 nucleotides, from 10 to 35 nucleotides, from 15 to 30 nucleotides, from 20 to 25 nucleotides, from 45 to 100 nucleotides, from 50 to 95 nucleotides, from 55 to 90 nucleotides, from 60 to 85 nucleotides, form 65 to 80 nucleotides, from 70 to 75 nucleotides) in length.

[0134] In yet other such aspects, a portion or all of the linker can include a polymer portion. For example, in some aspects, a portion or all of the linker can include a PEG-based portion. In yet still other such aspects, the linker can include a peptide portion and a nucleic acid portion, or a peptide portion and a polymer portion, or a nucleic acid portion and a polymer portion, or a peptide portion, a nucleic acid portion, and a polymer portion.

[0135] In some aspects, the nucleic acid-locked beacon can include one or more modifications. In some such aspects, the modifications make the beacon more stable and/or more resistant to degradation. In some aspects, the modifications can be chemical modifications, including but not limited to non-naturally occurring amino acids or nucleic acids.

[0136] In some aspects, the beacon can be barcoded. This is discussed in more detail below. [0137] The beacons disclosed herein can be combined with other structures which can aid in their delivery or ease-of-use. In some embodiments, the nucleic acid-locked beacon can be delivered via a single delivery vehicle. In some embodiments, the beacons may be delivered via one or more delivery vehicles each of a different composition. According to various embodiments, suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide- containing nanoliposomes, proteoliposomes, both natural and synthetically -derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphorsilicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domainblock polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic poly conjugates). [0138] In a specific example, the beacons can be coupled with a plasmonic nanostructure. Examples of plasmonic nanostructures which can be used with the beacons disclosed herein include, but are not limited to, those made from rhodium (Rh), platinum (Pt), gold (Au), or silver (Ag). In some aspects, the beacon can be associated with a nanostructure such as a nanoparticle or microparticle. In some such aspects, the nanoparticle can be a gold nanoparticle. In other such aspects, the microparticle can be a silica microparticle. In other aspects, the beacon can be associated with a plasmonic nanostructure as described in S. Pandit, et al. “DNA-Barcoded Plasmonic Nanostructures for Activity-Based Protease Sensing.” Angew. Chem. Int. Ed. 2024, 63, e202310964, which is hereby incorporated by reference in its entirety.

[0139] In some aspects, the signaling nucleic acid can be a single strand anti-sense DNA used for gene therapy. For example, the enzyme can interact with the beacon, which can then release the signaling nucleic acid. The signaling nucleic acid can then directly act as anti-sense DNA, or it can trigger an event which then leads to the release of anti-sense DNA which can be used for therapeutic purposes.

[0140] In some aspects, the enzyme can be a marker of cancer, inflammation, infection, virus, cardiovascular disease, or other disease or disorder.

[0141] In yet another aspect, provided is a diagnostic assay for determining presence of an enzyme, the assay including any of the disclosed beacons. The assay can further include other components which facilitate the detection or targeting of enzymes in vitro or in vivo.

METHODS

[0142] Contemplated herein is a method of detecting an enzyme. This can be done by determining the peptide target of the enzyme, then using all or a portion of that peptide with a beacon (DPLB) disclosed herein. [0143] In an aspect, provided is a method of detecting an enzyme of interest in a sample, the method including: a) exposing the enzyme of interest to a composition including a nucleic acid-locked beacon, wherein said nucleic acid-locked beacon includes: i) a cleavable domain; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary' to the signaling nucleic acid sequence; wherein said exposure can occur under conditions which can allow the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain can allow the signaling sequence to produce a detectable signal; and b) detecting the signal produced by the signaling nucleic acid sequence, thereby detecting the enzyme of interest.

[0144] When detecting an enzyme in vivo, the compositions disclosed herein can be used in a pharmaceutical carrier which allows for targeting to specific areas of the body, where detection of the enzyme may be desired. For example, the composition can be in a nanoparticle and can be directed toward a diseased area, such as cancer, or an area of inflammation. This information can then be used to target a specific treatment to that area. The beacon can also include a treatment modality, as described below.

[0145] In some aspects, steps a) and b) can further include: a) exposing two or more enzymes of interest to a composition including two or more nucleic acid-locked beacons, wherein each nucleic acid-locked beacon includes: i) a cleavable domain specific to one enzyme of interest; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure can occur under conditions which can allow each enzyme to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain can allow the signaling sequence to produce a detectable signal; and b) detecting the signals produced by each signaling nucleic acid sequence, thereby detecting each enzyme of interest. In some aspects, the two or more nucleic acid-locked beacons can allow for the detection of two or more different enzymes in the same assay (multiplex).

[0146] In another aspect, provided is a method of determining usefulness of a test compound in modulating an enzyme, the method including: a) exposing the test compound to the enzyme to form a test composition; b) exposing the test composition to a composition including a nucleic acid-locked beacon, wherein said nucleic acid-locked peptide includes: i) a cleavable domain; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure can occur under conditions which can allow the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain can allow the signaling sequence to produce a detectable signal; and c) detecting the signal produced by the signaling nucleic acid sequence, thereby detecting the enzyme of interest; and d) using said signal to determine an effect of the test compound on action of the enzyme. In some aspects, the effect of the test compound on action of the enzyme can include an increase in enzyme amount or activity, and wherein the signal may be greater than a reference signal produced by the enzyme not exposed to the test compound. In other aspects, the effect of the test compound on action of the enzyme can include a decrease in enzyme amount or activity, wherein the signal may be less than a reference signal produced by the enzyme not exposed to the test compound. This method can be carried out in vivo or in vitro.

[0147] In some aspects, steps a) and b) can further include: a) exposing the test compound to two or more enzymes of interest to form a test composition; b) exposing the test composition to a composition including two or more nucleic acid-locked beacons, wherein each nucleic acid-locked beacon includes: i) a cleavable domain specific to one enzyme of interest; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary’ to the signaling nucleic acid sequence; wherein said exposure can occur under conditions which allows each enzyme to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain can allow the signaling sequence to produce a detectable signal; and c) detecting the signals produced by each signaling nucleic acid sequence, thereby detecting each enzyme of interest; and d) using said signals to determine an effect of the test compound on action of each enzyme. In some aspects, the two or more nucleic acid-locked beacons can allow for the detection of two or more different enzymes in the same assay (multiplex), and the method can be used to determine the effect of the test compound on two or more enzymes of interest simultaneously.

[0148] In yet another aspect, provided is a composition detected by any of the disclosed methods.

[0149] In yet another aspect, provided is a method of treating and/or preventing a disease or disorder causing an increase of an enzyme in a subject, the method including: a) administering to the subject a composition including a nucleic acid-locked beacon, wherein said nucleic acid-locked beacon includes: i) a cleavable domain; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure can occur under conditions which can allow the enzy me to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain can allow the signaling sequence to produce a therapeutic response. [0150] The signaling sequence can provide a therapeutic response in a number of ways, including gene therapy, protein replacement therapy, regenerative medicine, vaccines, and cancer therapy. This can, in some aspects, be accomplished by using signaling nucleic acid sequences encoding for functional genes, therapeutic proteins, tissue promoters, antigens to stimulate immune responses, and genes to trigger cell death.

[0151] In some aspects, step a) can further include: a) administering to the subject a composition including two or more nucleic acid-locked beacons, wherein each nucleic acid- locked beacon includes: i) a cleavable domain specific to one enzy me; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure can occur under conditions which can allow each enzyme to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain can allow the signaling sequence to produce a therapeutic response. In some aspects, the two or more nucleic acid-locked beacons can allow for the therapeutic response to two or more different enzymes in the same subject.

[0152] The methods disclosed above can take place in vitro, in vivo, or ex vivo. When detection of an enzyme occurs in vitro, a variety of means can be deployed which allows for the detection of the signaling nucleic acid, which indicates the presence of an enzy me. This can be done using a nucleic acid amplification system, such as polymerase chain reaction (PCR), exponential amplification reaction (EXPAR), loop-mediated isothermal amplification (LAMP), next-generation sequencing (NGS), strand displacement amplification (SDA), or hybridization chain reaction, or using a PCR-free system. Systems which make use of anucleic acid amplification system can first amplify the signaling nucleic acid, then it can be detected using a variety of methods, including primer/probe recognition, sequencing, etc.

[0153] Also disclosed are non-PCR methods of detecting the signaling nucleic acid. Examples of non-PCR detection technology' can be found, for example, in Yang D, Yang L, Wang P. Nucleic Acid Molecular Systems for In vitro Detection of Biomolecules. ACS Mater Au. 2022 Nov 22;3(2): 83-87, herein incorporated by reference in its entirety'. One specific example is toehold-mediated strand displacement (TMSD) can also be used for detecting nucleic acids of specific sequences, where a single-stranded overhang serves as a toehold to initiate binding to target molecules which subsequently displaces the partially bound molecule. Strand displacement may also integrate with biochemical machineries for nucleic acid detection. For example, a molecularly triggerable riboswitch can be used to detect a target RNA molecule. The binding of target RNA onto the riboswitch recruits ribosome and initiates the synthesis of functional proteins that is compatible with fluorescent or colorimetric assays. [0154] The CRISPR-Cas system can also be used for nucleic acid detection. crRNA can specifically recognize and bind to signaling nucleic acid, and activate the nuclease activity of Cas proteins. In some such aspects, the detection system can include a Casl2a or Casl3a RNP system. In some such aspects, the signaling nucleic acid sequence can activate a Cas molecule, and the Cas molecule can cleave a nucleic acid reporter, thereby causing a fluorescence signal. For example, the signaling nucleic acid sequence can hybridize with the guide RNA complexed to an inactive Cas molecule. This can activate the Cas molecule and allow it to cleave a reporter. In some such aspects, the reporter can include a fluorophore and a quencher, wherein the quencher can prevent detection of the fluorophore when the reporter is uncleaved, and wherein the fluorophore can be detected when the reporter is cleaved by the activated Cas molecule. In some aspects, the reporter can be a DNase or RNase reporter. Cleavage of a nucleic acid reporter can lead to the output of signals that can then be detected.

[0155] The signaling nucleic acid molecules can also be barcoded, and the barcodes can be detected using the methods described herein and methods known to those of skill in the art, including sequencing methods.

[0156] The signaling nucleic acids can also be directly labeled with fluorescent molecules which can be detected. Examples of such assays include, but are not limited to, molecular beacons. The term “molecular beacon” refers a to detectable molecule, where the detectable property of the molecule is detectable only under certain specific conditions, thereby enabling it to function as a specific and informative signal. Non-limiting examples of detectable properties are optical properties, electrical properties, magnetic properties, chemical properties and time or speed through an opening of known size. An example is the FRET system.

[0157] The beacons disclosed herein can also be used in vivo. Nucleic acids, including the signaling nucleic acids described herein, can be detected in vivo in many ways, including, but not limited to, using nucleic acid biosensors. These biosensors can be used with technology to increase the intensity of signal output, the fluorescent reporter with longer emission wavelength, as w ell as magnetic resonance (MRI), positron emission tomography (PET), and photoacoustic (PA) signal were introduced to increase the tissue penetration. Other examples of means to detect nucleic acids in vivo include aptamers and DNAzymes as recognition groups, as well as molecular beacons, which are described above. Other methods can be found in Liu et al., Nucleic acid sensors in vivo: challenges and opportunities; View 29 March 2023, herein incorporated by reference in its entirety . EXAMPLES

Example 1: DNA-Locked Peptide Beacons for CRISPR-Amplified Sensing of Protease Activity

[0158] Proteases are a large family of enzymes that serve the fundamental role of catalyzing the breakdown of peptides and proteins in the human body [1]. In nearly all biological processes, ranging from cell differentiation, apoptosis, protein turnover, signal transduction, post translational modification, etc., the activity of proteases is necessary to occur [1] [2] [3], Along with the importance in healthy systems, dysregulations in protease activity have also been shown to be a key factor in a variety of diseases such as cancers, atherosclerosis, autoimmune disorders, Alzheimer's, heart diseases, and more [1] [4] [5] [6] [7] [8] [9], The sheer number of diseases which have been correlated to activity dysregulations has yielded the need to develop versatile detection platforms, in an effort to understand fundamental phenomena of the diseases, improved diagnostic tools, and activity-based therapeutics. Due to the -600 different potentially dysregulated proteases in the human body, their detection plays a pivotal role in the development of a broadly applicable diagnostic biomarker that could be instrumental in identifying and understanding a wide range of diseases.

[0159] In efforts to utilize proteases as disease biomarkers, traditional abundance-based testing has been routinely unable to provide a strong link between protease concentration and diseases [10] [11], As this is likely because the overall concentration of proteases is not characteristic of their overall activity, due to their expression as inactive zymogens, requiring bodily processes to activate them [10] [1 1], Therefore, techniques that focus solely on detecting active proteases, termed activity-based sensing, have come into recent interest [11] [12], Current methods testing specifically for protease activity include protease inhibitor-based probes that selectively bind to active proteases [13] [14] [15]. fluorophore quencher labeled peptide systems [12] [16] [17] [18] [19] [20] [21], mass spectrometry to detect peptide fragments [22] [23], and more. These techniques have demonstrated that activity-based protease sensing can show a significant correlation between disease presence and protease activities [10] [11], These techniques go on to show that activity-based protease sensing of diseases can even have the possibility for improvements in cancer detection over cunent methodology [12],

[0160] Current activity -based protease sensors face a number of fundamental limitations. These include the requirement of high probe concentrations, the absence of amplification handles (which severely limits their detection in samples of low protease concentrations), the requirement of lengthy or complex separation steps, or the necessity of sophisticated equipment. DNA-barcoded strategies, wherein peptides are conjugated to a DNA strands, can combine the advantages of having DNA (predictable and reconfigurable structures, signal transduction and amplification handles, and barcoding) [39] and peptides (programmability, recognition) have been used to alleviate a number of challenges current probes have faced. With these constructs, it has been shown that by pairing the protease-specific sensing capabilities of peptides with nucleic acids, strategies are capable of detecting protease activities with remarkable sensitivity and selectivity [26] [27], However, in existing DNA-barcoded systems, a separation step was necessary to separate intact DNA-barcoded peptides from those digested by the protease. This additional step added complexity and may result in losses of cleaved substrate. Moreover, in systems involving attachment of peptides to nanoparticle surfaces, steric effects can significantly restrict the ability of proteases to cleave the target peptides. This necessitates the use of longer peptides often necessary as short peptides are often not accessible.

[0161] To address these limitations, a study was conducted which introduces DNA-Locked Peptide Beacons (DLPBs), a DNA-based activity-based sensing strategy for activity-based protease detection. Unlike their predecessors. DNA-locked peptide beacons are not bound to separation moi eties but instead are free in solution and therefore require no separations, remove the need for sophisticated instrumentation by relying on commonly available fluorimeters for readouts, provide an amplifiable detection handle, and have quick assay response, while not sacrificing the sensitivity or selectivity of previous methods. The incorporation and detection of the DNA handle is a vast improvement upon previous locked hairpin detection platforms, allowing for versatility in both design and detection [40] [41], These molecular constructs are designed to snap into a ‘locked” form restricting the ability of the DNA handle for signaling. Upon recognition with a specific protease, the structure is split, resulting in a spontaneous change into an “unlocked” signal transducing state, which in this study was detected using a CRISPR-Casl2a enzymatic detection platform. Casl2a is a protein in the CRISPR family which functions as an 'activatable enzy me' [42], In solution CRISPR-Casl2a complexes with a guide RNA sequence, causing the forming a ribonuclear protein (RNP), in an inactive state. The RNP can then become selectively activated by hybridization of a DNA complementary to the guide RNA. This activated state endows Cast 2a with a single-stranded DNA (ssDNA) nuclease activity, that when combined with a fluorophore-quencher labeled DNase reporter, provides an enzy matically generated fluorescence signal. Activity of the CRISPR-Casl2a detection platform were also tested at a variety of concentrations. RNP, and with the presence of proteases, to optimize the system. Crucially, the use of CRISPR-Casl2a in the method streamlines the process of protease activity detection into a two-step, one-pot reaction, which can be completed within 45 minutes, significantly simplifying the detection of protease activity, making it significantly more practical to observe protease activity.

Results and Discussion

[0162] DLPBs are composed of three key components: (1) a peptide domain that is recognized and cleaved by the protease of interest, (2) a signaling DNA sequence that is complementary and can activate the RNP system, and (3) a short “blocking’⁷ DNA to create the locked form. The blocking DNA is complementary to a portion of the initiator strand such that it forces them to hybridize to adopt a locked hairpin-like conformation (see all RNA and DNA sequences used in this study in the supplemental information TABLE 2 and TABLE 3 respectively). It was hypothesized that the presence of the peptide in the hairpin loop and the partial blocking of the initiator sequence would sterically prevent the RNP complexes from binding to the initiator sequence while in the locked state. 3D modeling of DLPBs and RNPs show they are of comparable size. When exposed to a specific protease, the peptide region is recognized and cleaved (FIG. 1A). Consequently, the probe transitions from an intramolecular DNA duplex to an intermolecular DNA duplex, with concomitant lowering of the melting temperature (T_m) of the duplex. The locked form has a T_m above room temperature (RT) and therefore forms the hairpin structure, however after cleavage the unlocked form has a T_m lower than RT. Therefore, at ambient conditions the blocking DNA is forced to dissociate from the activator strand, causing the unlocking of the activator strand’s function. This unlocked signaling DNA can then activate the CRISPR-Cas l2a enzymes, generating an amplified fluorescence signal (FIG. IB). The activation of CRISPR-Casl2a enzymes with DNA hairpins was also investigated, as well as blocked double stranded DNA structures, with further hypothesis of how CRIPSR-Casl2a systems interact with other locked DNA structures.

[0163] The study first determined the desirable length of the blocking DNA computationally using the Integrated DNA Technologies’ OligoAnalyzer™ tool. The criteria for selecting an ideal blocking strand w as a sequence that (i) has a T_m above room temperature in hairpin form, (ii) but has a T_m below RT as an intermolecular duplex, and (iii) allows the probe to maintain hairpin structures under a range of ionic strengths encompassing biologically relevant conditions. By examining the T_m for blocking DNA of different lengths (2 to 13-nt) at varying ionic strengths (10-300 mM NaCl and 0-40 mM MgCb) and varying hairpin loop sizes (10,30 nt). It was determined that at InM DLPBs, with 10-30 nt loops, 100 mM NaCl and 40 mM MgCh, an 8-nt blocking DNA would be ideal, as it would form a hairpin structure at 25°C, but also spontaneously separate into ssDNA after proteolytic cleavage. [0164] As a proof-of-concept, the study created a DLPB for detecting the 3-chymotrypsin- like main protease (3CL) of SARS-CoV-2. 3CL was chosen as the model protease of this system due to its relevance in the detection of active SARS-CoV-2 infections, as well as its well-defined substrate. The peptide sequence, CSAVLQ|SGFK(Ns) (SEQ ID NO: 1, where J, indicates the cleavage site), was modeled after commercial 3 CL peptide reporters and was modified to include an azide modified lysine at the C-terminus (see TABLE 1 for all peptide sequences used in this study). Using this peptide, DLPBs were synthesized. The DNA and peptide sequences were synthesized using solid phase phosphoramidite and peptide synthesis. The initiator sequence contained a DBCO modification on the DNA’s 5’ end for subsequent attachment to the peptide via Cu-free click chemistry. Subsequently, a second azide group was installed on the N-terminus of the peptide through NHS-ester chemistry. This then under-went a second reaction of Cu-free click chemistry to a 3’ DBCO modified blocking strand. DNA concentration, in-corporation of modifications, and completion of copper-free click chemistry was validated using UV-Vis spectrometry'. The solution was then purified via HPLC or gel electrophoresis to yield the final DLPB, and characterized using MALDI-mass spectrometry and gel electrophoresis.

[0165] The DLPB structure and the ability of proteases to cleave DPLPB structures was confirmed using mass spectrometry (FIG. 2A). To do so, the 3CL DLPB was treated with 3CL protease overnight and then the solution was analyzed to reveal that the parent DLPB peak disappears, and the appearance of 2 peaks that correlate to the expected proteolytic fragments appear. This indicated that the DLPB structure can be cleaved in the peptide region. The hairpin-like structure of the DLPBs was confirmed using melting temperature analysis of a fluorophore quencher labeled DLPB, that when dehybridized provides a fluorescence signal (FIG. 2B). Upon conjugation of the blocking and signaling strands the T_m increases significantly and has the approximate T_m of a DNA hairpin of the same size. This indicated that the DLPB synthesis was successful, as well as that the DLPBs are forming the locked structure in solution. The FQ 3CL DLPB was then treated with 3 CL protease, and the Tm was measured. After treatment with the 3CL protease, the T_m shifted to what is observed for intermolecular DNA hybridization, indicating that after the 3CL cleavage the DLPB transitions into the unlocked ssDNA structure. To benchmark the ability of DLPBs, they were compared to commercial FQ probes as well as synthesized FQ based DLBPs. A FQ based DLPB was also synthesized to evaluate the extent of signal amplification provided by the CRISPR-Casl2a assay. By incubating FQ hairpin with 3CL protease the resultant cleavage of the peptide separates the fluorophore and quencher, causing an increase in fluorescence signal, without amplification. This ability was shown in a time dependent assay in which 100 nM of the FQ DLPB was treated with 1 pM 3CL protease and the resultant cleavage generated significant florescence over time. When comparing the DLPBs with the commercial FQ substrates and FQ RNP 3CL DLPBs, significant advantageous of using the DLPBs were showcased. To test the DLPB efficacy in detecting protease activity', 10 nM of the 3CL-specific DLPB (3CL DLPB) was incubated with 500 pM of the 3CL protease for 30 minutes in a commercial 3CL buffer, facilitating 3CL-mediated cleavage of the peptide substrate to releasing the signaling strand (FIG. 2C). Subsequently, this solution was incorporated with 10 nM of the CRISPR-Casl2a RNP complex along with a DNase fluorogenic substrate, maintaining a final concentration of 1 nM DLPB. The released signaling DNA could then complex with the RNP, where uncleaved DLPBs in their inactive hairpin conformation are unable to, activating it. This allows the CRISPR-Casl2a enzymes to cleave the DNase substrate thereby generating an amplified fluorescence signal that was read via a plate reader. This was also run with RNP only and no protease DLPB controls. Within 15 minutes, there was a discernable increase in fluorescence compared to the control. After 90 minutes, the relative signal of the DLPB with 3CL protease was upwards of 100-fold greater than a DLPB without the 3CL protease treatment. This procedure was also altered to allow for a one-pot detection of protease activity. DLPBs were also tested before and after purification and at a variety of different concentrations and temperatures.

[0166] When the commercial 3CL substrate was incubated at the same concentration as the DLPB with 3CL protease, no significant fluorescence enhancement was observed (FIG. 2D). Even when using concentrations of the commercial probe 100-fold greater than DLPBs, it generated a signal 1.4-fold less than the DLPB system. Using the same procedure, the FQ DLPBs were incubating 100 nM of the FQ hairpin with 250 nM and 500 nM of 3CL protease, a 1.6-fold and a 2.6-fold increase in signal was generated over the control. Since even at 100- fold lower concentrations a 2.6-fold increase of 3CL DLPB over FQ 3CL DLPB and a 1.4-fold increase over commercial probes, indicates the sensitivity' advantage of using CRISPR based amplification. These studies underscore that DLPBs can effectively detect protease activity in conjunction with CRISPR-Cas technology and highlights the system's superiority in comparison to both commercial and FQ-based methodologies.

[0167] To assess the versatility of DLPBs, multiple probes were synthesized utilizing peptide sequences tailored for proteases such as cathepsin B (CTSB), matrix-metalloprotease 7 (MMP7). and caspase-3 (CASP3) (FIG. 3 A). When incubated with their respective proteases, a pronounced increase in fluorescence signal yvas observed, indicating the successful detection of the target proteases. (FIG. 3B) The ability to detect these specific proteases is of high physiological relevance, as they are involved in various diseases and with a high association of dysregulation in cancers. In cancer, CTSB is implicated in tumor progression and metastasis, MMP7 in tissue remodeling and cancer, and CASP3 in apoptosis, making their detection crucial for fundamental understanding and potential diagnostics platforms. Importantly, because the DLPBs can be readily adapted to sense for different target proteases, they represent a versatile and powerful tool that can be tailored for the detection of other relevant proteases as well, promising broad applicability in disease diagnosis and therapeutic monitoring.

[0168] The study next evaluated if DLPBs could further be used for detection of diagnostically relevant protease activity by focusing on CTSB due to its overexpression in various cancers. To determine the limit of detection (LOD) of CTSB DLPB in buffer, a calibration curve was created by incubating 10 nM CTSB DLPB with decreasing concentrations (20-1 nM) of CTSB protease. Following this, the hairpin was added to 10 nM RNP to achieve a final concentration of InM, and fluorescence was measured (FIG. 4A). At 60 minutes, the response was plotted relative to CTSB concentration which yielded a linear fit (FIG. 4B). The LOD was determined to be as low as -146 pM. significantly surpassing the capabilities of commercial probes which were unable to operate at similar conditions. The LOD was also investigated for the 3CL protease. This sensitivity⁷, without the need for high concentrations of DLPBs, indicates potential relevance for single cell analysis, targeted tissue diagnostics, and personalized therapy monitoring, where large probe quantities are impractical. The selectivity of DNA-locked peptide beacons was further investigated by testing the CTSB- specific hairpin against a variety' of proteins and proteases, including 3CL, MMP7, thrombin, caspase-3, try psin, and bovine serum albumin (FIG. 4C). The CTSB DLPB was incubated with 20 nM of each protein for 30 minutes and was then added to the RNP and the fluorescence measured. The CTSB DLPB had meager enhancements for all non-specific species, -1-2.3- fold enhancement, save for MMP7 protease where the enhancement was 2.6-fold. Compared to the probe it showed 13.0-4.8-fold more selectivity for CTSB than for any other test-ed species, highlighting its specificity even when tested against other proteases.

[0169] The study' next evaluated the use of these probes in biologically relevant samples by treating them with cellular lysates from patient-derived colon cancer cells. It has been shown previously that levels of cathepsin B activity' are significantly heightened in these cells, making them a prime target for detection using DLPBs [35] [36] [37] [38], After lysing the cells and extracting them into PBS, the lysates were incubated with CTSB DLPBs for 30 minutes and then introduced to the RNP and the fluorescence measured (FIGS. 5A-5D). Upon treating the DLPBs with HT29, NCI-H508, RKO, and SW-620 cell line lysates, enhancements of 3.6, 4.8, 6.0, 8.6, and 16.4-fold was observed respectively after 30 minutes. The other DPBPs (MMP7 and CASP3) were also treated with the cell lysates. In using the most responsive line, SW-620, the probe was successfully used to detect cathepsin B activity from as few as 6,000 cells. A commercial probe was also employed, which supported the presence of CTSB in the lysates. As an additional control. 3CL DLPBs were also incubated with the cellular lysates to evaluate any background effects on the system. Since there is no expected 3CL protease concentration in colon cancer samples, any significant signal enhancement could be from disruptions of the locked structure or non-specific protease activity. In incubating the 3CL DLPB w ith the cellular lysates, enhancements ranged from 1.7-3.7-fold, with CTSB DLPBs providing enhanced signals compared to this control, supporting the successful identification of CTSB in human- derived samples. This also served to validate the presence of active proteases in complex media through the detection of spiked 3CL protease in cell lysates. Furthermore, to ensure the activity detected was indeed from active CTSB, a commercial CTSB inhibitor was added to the lysates, leading to the response to drop back to controls of the commercial probe and DLPB minus protease incubation (FIGS. 5E-5F). Overall, these results support the successful identification of CTSB activity⁷ in cancer cell samples, suggesting that these probes are effective in complex biological media and may be valuable for diagnostic applications.

[0170] In conclusion, this study has developed an innovative sensing methodology by using DLPBs to leverage detectable protease-induced conformational transformations. The ability of the DNA to hybridize into a locked, inert signaling state bypasses the prior need of separation in nanoparticle-based platforms. The confirmational change brought about by proteolytic cleavage to unlock a strand of signaling DNA is a method to generate a handle for amplification and signaling and could be utilized for other activity -based sensing platforms and could be paired with a myriad of DNA based detection platforms with the correct choice of the blocking strands. Longer blocking strands could be utilized to perform detection with DLPBs at elevated temperatures or conditions which impact the systems melting temperature. Using CRISPR-Casl2a-mediated signaling, DLPBs offer a simple, room temperature, interchangeable, and amplifiable method to detect protease activity. DLPBs offer a limit of detection that holds immense promise at such low probe concentrations for use in detection where use of high peptide concentration is suboptimal. The utilization of standard fluorimeters for readouts, combined with a swift assay response, augments the potential applications of this technique for benchtop analysis better than conventional methods with an almost 150-fold enhancement in sensitivity when corrected for concentration. Moreover, by tailoring the peptide sequences within the DLPB design, it is shown that DLPBs can be programmed to detect a number of different proteases with the possibility to be utilized for a vast number of different targets. The study also reveals a profound sensitivity towards cancer-related proteases like CTSB, even when used in complex biological matrices. The structure of DLPBs, by facilitating a locked/unlocked transition, not only lays a robust foundation for detecting proteases, but also opens avenues for detecting other catalytic enzymes. Cumulatively, the flexibility, specificity, and sensitivity of this strategy positions DLPBs as a versatile tool, poised for further use in the field of activity -based detection.

[0171] TABLE 1, TABLE 2, and TABLE 3 give the peptide and DNA sequences used in this study.

TABLE 1. Peptide sequences

TABLE 2. Guide RNA sequences used in this study. The part of the gRNA sequence that is complementary to the activating sequence is in bold. TABLE 3. DNA sequences.

[0172] Modeled CRISPR-Casl2a, DNA Hairpins, and DLPBs: This study used CRISPR-Casl2a in complex with gRNA and DNA activator. FIG. 6A depicts DNA hairpin 30-8 sequence modeled from TABLE 3 hairpin using Integrated Technologies OligoAnalyzer tool. FIG. 6B depicts DNA hairpin 12-8 sequence modeled from TABLE 3 hairpin using Integrated Technologies OligoAnalyzer tool. FIG. 6C depicts DNA non-hairpin 12-10 sequence modeled from TABLE 3 hairpin using Integrated Technologies OligoAnalyzer tool. FIG. 6D depicts a 3D ball and stick representation of the 3CL DLPB was created using Avogadro and ChemDraw then modeled using PyMOL A size comparison between DLPBs and CRISPR-Casl2a enzymes was completed to visualize what effect the size of the DLPBs would play on the restriction of activation via CRISPR-Casl2a. which is shown in FIG. 6E. [0173] It is hypothesized that the conformation of DLPBs should restrict the activation of CRISPR-Casl2a until cleaved by an active protease. Uncleaved DLPB should be restricted from activating CRISPR-Casl2a for two reasons: 1) the presence of a blocking sequence that competes with the hybridization of the activating sequence and the gRNA, and 2) a steric hindrance between the loop region of the DLPB and the CRISPR-Casl2a enzyme. After DLPBs are cleaved by target proteases, the resultant single stranded activator DNA should have neither of these issues and activate CRISPR-Casl2a less impeded. Therefore, CRISPR- Casl2a enzymes could discern this conformational change and go on to provide an amplified florescence signal as a proxy for the activity of select proteases. This hypothesized interaction is shown in FIGS. 7A-7B.

Materials and Methods

[0174] DNA Synthesis and Materials: DNA sequences were synthesized using solid phase DNA synthesis using a MerMade 6 automated DNA synthesizer (LGC Biosearch Technologies Alexandria MN 56308). DNA was synthesized on solid support universal controlled pore glass (CPG) beads (Glen Research, Uny Support CPG-1000, Cat. No. 20-5040), or on 3’ black hole quencher CPG (3 -BHQ-2 CPG, Cat. No: 20-5932-01) in the case of the Quencher-labeled Activator DNA. After synthesis the beads were treated with 1 mL/1 LLM DNA, of 30% ammonium hydroxide solution (Sigma-Aldrich, Cat. No. 221228-100ML-A) for 17 hours at room temperature (RT). The solution was then evaporated under pressurized air at RT until the ammonium hydroxide had evaporated, approx. 10 minutes. The DNA solution was then separated from the beads using a filter syringe and was later purified and characterized depending on the sequence. All DNA phosphoramidites and materials required for solid phase DNA synthesis were purchased from Glen Research (dA-CE phosphoramidite, Cat. No: 10- 1000-10), (dC-CE phosphoramidite, Cat. No: 10-1010-10), (dT-CE phosphoramidite, Cat. No: 10-1030-10). (DMF-dG phosphoramidite, Cat. No: 10-29-10), (DBCO-dT-CE phosphoramidite. Cat. No: 10-1539-95), (5 ’-DBCO-TEG Phosphoramidite, Cat. No: 10-1941- 90), (Cyanine 5 Phosphoramidite, 10-5915-95), (0.25M 5-Ethylthio-lH-Tetrazole (ETT) in Anhydrous Acetonitrile, Cat. No: 30-3140-52), (3% TCA/DCM, Cat. No: 40-4140-57), (0.02M Iodine in Tetrahydrofuran/Pyridine/Water (70:20: 10). Cat. No: 40-4330-52), (Anhydrous acetonitrile, 40-4050-57). Sequences made this way include activating DNA. 8-nt Blocking DNA, Quencher-labeled Activator DNA, Dye-labeled Blocking DNA, CRISPR-Casl2a activator. Sequences purchased custom from Integrated DNA Technologies include DNA Hairpin 30-8, DNA Hairpin 12-8, DNA Hairpin 12-0, 24-nt DNA blocking DNA, and 16-nt DNA blocking DNA.

[0175] DNA Purification and Characterization: Unmodified DNA sequences or those containing 5 ’DBCO-TEG groups, were purified using a Glen-Pak cartridge (Glen Research, Glen-Pak DNA purification cartridge. Cat. No. 60-5200) following the manufacturer’s recommended protocol. DNA containing a 3’DBCO-dT group, specifically the 8-nt Blocking DNA, was purified through high performance liquid chromatography (HPLC) using a Vanquish HPLC (ThermoFisher Scientific, Vanquish Core HPLC Systems, Cat No: VQ- CORE-BIN-01). Buffer A was 30 mM triethylammonium acetate (prepared by mixing 1 :1 molar equivalents of triethylamine (Fisher Scientific, Triethylamine (Reagent), Cat No: 04885-1) and glacial acetic acid (Fisher Scientific. Acetic Acid, Glacial (TraceMetal Grade). Cat No: A507-P500) over ice to pH 7 with 3% acetonitrile (ACN) in water, while buffer B was 100% acetonitrile. The method followed a gradient increase from 10% to 35% buffer B over 80 minutes, a ramp to 100% buffer B over 5 minutes followed by 5 minutes of 100% buffer B, a ramp down to 5% buffer B over five minutes followed by 5% buffer B for 5 minutes. Separation was monitored through UV-Vis peaks at 260 nM and 310 nM for DNA and DBCO respectively. All peaks were collected, lyophilized, and redissolved in water for characterization.

[0176] Peptide Synthesis, Purification, and Characterization: All peptides were synthesized in house using standard Fmoc-based solid phase synthesis on a 0.1 mmol scale. Synthesis was conducted using Rink Amide AM resin 100-200 mesh (Sigma- Aldrich, Cat. No. 183599-10-2) inside a fritted 6 mL syringe. The resin was initially prepared by washing with 5 mL of dimethyl formamide (DMF) (Millipore Sigma. N.N-Dimethylformamide. Cat No: 270547), for 15 minutes. Peptides were synthesized by adding amino acids sequentially in the follow ing cycle: the functionalized resin was washed with 5 mL of dimethyl formamide (DMF) followed by deprotection using 5 mL of 20% piperidine (Millipore Sigma, Piperidine Solution 20% in DMF, Cat No: 80645) in DMF for 5 minutes then the same reagent for a further 20 minutes. Piperidine was washed from the syringe using DMF until the piperidine smell disappeared. The amino acid was then added to the resin along with coupling reagents (2.5 equiv. HOBt, 2.5 equiv. DIC) in 3 mL DMF. This coupling w as run at 2 hours RT but couplings involving Fmoc Lys (N3)-OH (AnaSpec.Inc. Cat. No. AS-53100-F1) were allowed to run overnight. This cycle was follow ed until all amino acids had been added and then deprotected using the earlier mentioned procedure. Peptides were cleaved from the resin surface using 1 mL of a cocktail solution of trifluoroacetic acid (TFA) (Sigma- Aldrich, Cat. No. 302031- 100ML), H2O, and triisopropylsilane (TCI, Cat. No. T1533) in a volumetric ratio of 95:2.5:2.5, shaking at RT for two hours. Taking 100 pL portions of this solution and adding them dropwise into chilled diethyl ether (Millipore Sigma, Diethyl ethyl, Cat No:296082) the peptide was precipitated and recovered using centrifugation at 15,000 rpm for 5 minutes. This was repeated until all the solution was collected and was further washed using this method using fresh chilled diethyl ether. The peptide was then stored dry at 4°C until further use.

[0177] Peptide-DNA Conjugates Synthesis, Purification, and Characterization: Peptide-DNA conjugates were synthesized through copper free click chemistry between azide conjugated peptides and dibenzocyclooctyne (DBCO) modified DNA. The peptides used were (Pep-3CL, Pep-MMP7, Pep-CTSB, and Pep 3 from TABLE 1), and the DBCO DNA was the activator DNA as seen in TABLE 3. DNA-Peptide conjugation through click chemistry was carried out at a 5 : 1 molar ratio in respect to the peptide to DNA in lx phosphate buffered saline (PBS) (Thermo scientific, pH 7.4, Cat. No: J62036.K2) shaking at RT overnight. For downward synthesis of DLPBs, 1 pmol of activating DNA w as used and reacted at approx. 600 pM. The reaction was monitored for completion by the disappearance of the DBCO’s characteristic 310 nm peak via UV-Vis. The solution was then washed using a 3K centrifuge filter (Amicon Ultra, UFC500396) five times with 0.5 mL of ultrapure water and conjugation was characterized via UV-Vis.

[0178] The retained portion was then purified using a Macro-Prep DEAE weak anionic exchange resin (Bio-Rad, Cat. No: 158-0020). Into 2 fritted 6mL syringes, 2 mL of the DEAE resin was loaded and washed with 5 column volumes of ultrapure water. Before applying the sample, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Sigma Aldrich, Tris(2- carboxyethyl) phosphine hydrochloride. Cat No: C4706) was added to a final concentration of 1 OmM and allowed to shake at 1500 RPM (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) for 15 mins to break any disulfide bonds. The entirety of the sample was then applied to the DEAE at a flow rate of 1 drop per second, washed with 5 column volumes of water, then 0.3 M NaCl, and peptide-DNA conjugated were then eluted with 1 mL of 1 M sodium chloride (NaCl) (Fisher Bioreagents, Sodium Chloride, BP358-212). The elution of the DNA was monitored using UV-Vis and DNA characteristic absorbance at 260 nM. No significant DNA peaks were seen in water or 0.3M NaCl washes while a large portion (approx. 50%) of the expected DNA was recovered using 1 mL of 1 M NaCl. Further washing with additional 1 M NaCl or higher concentrations did not yield additional DNA. The sample was then washed using a 3K centrifuge filter five times with 0.5 mL of ultrapure water and conjugation was characterized via UV-Vis.

[0179] Linker-Peptide-DNA Synthesis, Purification, and Characterization: Linker- Peptide-DNAs were synthesized though NHS-ester conjugation between peptide-DNA and NHS-PEG4- Azide (ThermoFisher, Cat. No: 26130). Before reacting, the peptide-DNAs were treated with TCEP at a final concentration of 10 mM shaking for 15 minutes. NHS-ester conjugation was carried out using 0.5 pmol of the peptide-DNAs and a 150-molar excess of the NHS-PEG4-Azides in 2 mL of sodium bicarbonate (Fisher Bioreagents, Sodium Bicarbonate, Cat No: BP328-500) shaking at RT overnight. The sample was then washed using a 3K centrifuge filter five times with 0.5 mL of ultrapure water and conjugation was characterized via UV-Vis. Final yield was on average was approx. 85%.

[0180] DLPB Synthesis: DLPBs were synthesized by reacting linker-peptide-DNAs with 8-nt Blocking DNA using copper free click chemistry. Blocking strands were kept at a 5: 1 molar excess to the linker-peptide-DNA strands. Before conjugation, the linker-peptide-DNAs were treated with afinal concentration of lOmM TCEP while shaking for 15 minutes. Then 0.1 pmol of the treated sample was conjugated to the blocking strand (5 : 1 molar excess of blocking strand to linker-peptide-DNA) in 0. 1 M NaCl PBS while shaking at 1500 RPM RT overnight to 48 hours. The sample was then washed using a 3K centrifuge filter ten times with 0.5 mL of ultrapure water and conjugation success was characterized via UV-Vis and MALDI-MS.

[0181] DLPBs Purification, and Characterization: Purification of DLPBs was accomplished using HPLC separation. Purification of 3CL DLPB, matrix metalloproteinase-7 (MMP7) DLPB, and cathepsin B (CTSB) DLPBs were accomplished using the following method. Buffer A was 30 mM triethylammonium acetate buffer pH 7 and 3% acetonitrile (ACN) in water, while buffer B was 100% acetonitrile. The method followed a gradient increase from 10% to 25% buffer B over 80 minutes, a ramp to 100% buffer B over 5 minutes followed by 5 minutes of 100% buffer B, then a ramp down to 5% buffer B over five minutes followed by 5% buffer B for 5 minutes. For the purification of the caspase-3 (CASP3) DLPB, a similar method was used but the gradient initially followed a gradient increase from 13% to 27% buffer B over 80 minutes. All other steps are kept the same as previously mentioned. All peaks were collected, lyophilized, and redissolved in water for characterization via MALDI- MS.

[0182] Fluorophore-Quencher 3CL DLPB, Synthesis and Characterization: Synthesis of a fluorophore-quencher (FQ) 3CL DLPB was accomplished using a similar procedure to regular DLPBs, with differences in the synthesis of the FQ 3CL DLPB described herein. In place of the activating DNA, a Quencher-labeled Activator DNA containing a 3’ black hole quencher was used, and the blocking strand was swapped for the Dye-labeled Blocking DNA with a 5’ cyanine 5 dye as described in TABLE 3. Care was taken to ensure the materials were stored in dark conditions as w ell as covered during handling to decrease the chance of them being damaged by stray light. All conjugation chemistry was the same as DLPBs except that the conjugation of linker-peptide Quencher-labeled Activator DNA to Dye-labeled Blocking DNA was done over 72H in a 1 : 1 ratio. After conjugation it was then washed in a 3K centrifuge filter 10 times with 0.5 mL of ultrapure water and characterized via MALDI-MS.

[0183] CRISPR Studies with DNA Hairpins: Preliminary testing of DNA hairpins was conducted to determine the effect of the absence of the sensing peptide on the activation of CRISPR-Casl2a assays. To closely mimic the structure of DLPBs, the peptide length was replaced with a comparable length of DNA bases. The contour length of amino acids ranges from 0.36 nm to 0.4 nm, while the peg linker's length is approximately 1.4 nm. 1 Thus, the estimated length of the peptide sequences varies betw een 5 to 5.4 nm. Given that the length of ssDNA bases is reported to be between 0.3 nm and 0.6 nm [2-4], the peptide region can accurately be substituted with 9-18 DNA bases. The study tested DNA hairpins of 12 bases and 30 bases. The use of a 30-mer DNA was chosen to ensure a broad enough range to observe distinct variations in CRISPR signaling and to provide a better representation of potential structural variations which may be hidden with only slight changes in the loop structure.

[0184] MALDI-MS Characterization: MALDI-MS characterization of DNA, peptides, peptide-DNA conjugates, linker-peptide-DNAs, and DLPBs was accomplished using aBruker autoflex maX MALDI-TOF/TOF instrument. The matrix utilized for all samples was prepared using 2',6'-Dihydroxyacetophenone (DHAP) (Sigma-Aldrich, Cat. No. 37468). The matrix was prepared by dissolving 25 mg of the DHAP in 333 pL of methanol (Fisher Chemical, Methanol (HPLC Grade), Cat No: A452Sk4) to which 111 pL of saturated ammonium citrate (Sigma Aldrich, Ammonium Citrate Dibasic, Cat No: 247561) was added dropwise. A white precipitate was immediately seen and allowed to settle for 15 minutes resulting in a clear yellow liquid layer which was used as the matrix. When plating for MALDI-MA, 2 pL of sample was added followed by 1 pL of the DHAP matrix. This resulted in rapid crystallization and was allowed to air dry, approx. 2 minutes, before MALDI-MS.

[0185] Confirmation of DLPB Synthesis: Using a MALDI-MA procedure, MALDI-MS of DBCO-DNA, Peptide-DNA conjugates, Linker peptide-DNA conjugates, and DLPBs MALDI-MS was completed to support successful synthesis of all intermediates and of final DLPBs.

[0186] MALDI-MS Confirmation of DLPB Cleavage: To verify the capability of proteases to cleave synthetic DLPB structures, MALDI-MS was employed both before and after treating DLPBs with protease. To examine the protease cleavage of DLPBs, 1 pM of the 3CL DLPB underwent treatment with 2 pM 3CL protease (Sigma Aldrich, Cat. No: SAE0172- 200UG). This process took place in a total volume of 100 pL 3CL Protease Assay Buffer (BPS Bioscience, Cat. No: 79956), with shaking at 1500 rpm at RT overnight. Subsequently, the solution was washed ten times using a 3K centrifugation filter with 0.5 mL of ultrapure water. The remaining fraction (approximately 10 uL) was characterized via MALDI-MS.

[0187] Fluorescence Assays

[0188] CRISPR-Casl2a DNA and DLPB Activity Assay Procedure. CRISPR-Casl2a ribonucleoprotein solution (RNP) was formed using CRISPR-Casl2a enzyme Alt-R™ L.b. CasI2a (Cpfl) Ultra (Integrated DNA Technologies, Ref. No: 443255472). guide RNA purchased from Integrated DNA Technologies (TABLE 2), and reporter DNase alert (Integrated DNA Technologies, Cat. No: 11-04-03-03). These were prepared in a CRISPR- Casl2a assay buffer (40 mM Tris-HCL pH 7.5, Invitrogen, Cat. No: 15567-027), (100 mM sodium chloride, Fisher Bioreagents. Cat. No: BP358-212), (40 mM magnesium chloride, Fisher Bioreagents, Cat. No: M33-500) (CRISPR-Cas 12a assay buffer). To ensure repeatability of CRISPR assays, stock guide RNA and CRISPR-Casl2a were aliquoted into single use tubes. Guide RNA was used directly while CRISPR-Casl2a enzyme was further diluted to 6.7 pM just before use with CRISPR-Casl2a assay buffer.

[0189] For activity assays, DNA or DLPBs were incubated with proteases in a total of 40 pL of assay buffer correlated to each protease. Samples are then incubated for 30 minutes to 24 hours shaking at 1500 RPM on a shaker (Benchmark. Multi-Therm Heating Shaker, Cat No: H5000-H) at 25°C. Controls were created and incubated in the same way however an equal amount of buffer was substituted instead of the protease. After this incubation was complete the solution could then be directly added to the activity assay. Note that all DNA or DLPBs are incubated at 10 x their final run concentration, with DLPB incubations occurring at 10 nM while the final run concentration is diluted to 1 nM.

[0190] To prepare the RNP for the activity’ assay, a solution of 20 nM CRISPR-Casl2a, 20 nM gRNA, and 5.0 uL/well of DNase alert was prepared in CRISPR-Casl2a assay buffer. When testing DNA or DLPBs, solutions are added to each well in a black 96 well plate in the following order, 40 pL of CRISPR-Casl2a assay buffer, 50 pL of 20 nM RNP 5.0 uL/well DNase solution, and 10 pL of the DNA or DLPBs samples. In 100 pL this gives a final concentration of RNP solution (lOnM CRISPR-Casl2a, lOnM gRNA, 2.5uL/well DNase alert) and DNA or DLPBs (InM) in a 10: 1 ratio. Unless otherwise noted, the RNP/DNase alert solution is added to all wells in CRISPR assays. Using a plate reader (Agilent, BioTek Cytation 5 Imaging Multimode Reader) samples are read at 25°C shaking for 30 seconds before reading fluorescence every three minutes (select assays were also run reading florescence instead every 5 minutes) at an excitation wavelength of 525 nm and an emission at 566 nm. A second plate reader was also used for CRISPR-Casl2a assays (Agilent, BioTek Synergy' Hl Multimode Reader), and it should be noted that the gain between these plate readers is different and therefore RFU signals may differ between experiments. Unless otherwise stated all CRISPR data was collected using these plate readers with these settings.

[0191] Modified One Pot DLPB CRISPR-Casl2a Activity Assay Procedure'. A second method using CRISPR-Casl2a, and protease incubated DLPBs was also achieved using a one pot reaction. For the detection of 3CL protease activity, 10 nM of 3CL DLPB was incubated with 1 pM of 3CL protease in 40 pL of 3CL buffer for 30 minutes to allow for proteolytic cleavage. This solution was then diluted with 180 pL of CRISPR buffer followed by 225 pL of 20 nM RNP solution. This gave a final concentration of 1 nM DLPB, 10 nM RNP, and 2.5 pL DNase alert/well. Into 4 wells, lOOuL of this solution was then added to a black bottomed 96 well plate and read. Controls of no DLPB (RNP and DNase alert solution) and DLPB with no protease were also run, with the volumes required substituted with equal amounts of buffer.

[0192] Comparison of 3CL DLPB and with Commercial 3CL Fluorogenic Substrate'. As a benchmark of the 3CL DLPB platform a commercially available 3CL fluorogenic substrate (Bio-Techne, SARS CoV-2 3CL protease substrate RHl lO-conjugated, Cat. No: S-720-200) was compared to the DLPBs using the same concentration of the DLPB at 1 OnM and at the manufacturers recommended concentration of 1 pM. After incubation the samples were diluted in 90 pL of CRISPR-Casl2a Assay buffer and read at Ex: 485 nm Em: 535 nm.

[0193] Comparison of CTSB DLPB with Commercial CTSB Fluorogenic Substrate'. As a benchmark of the CTSB DLPB platform a commercially available CTSB fluorogenic substrate (Sigma- Aldrich, Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride, Cat. No. C5429) was compared to the DLPBs using the same concentration of the DLPB at lOnM, and at the manufacturer recommended concentration of IpM. After incubation the samples were diluted in 90 pL of CRISPR-Casl2a Assay buffer and read at Ex: 380 nm Em: 460 nm.

[0194] Detection of CTSB, MMP7, 3CL, and CASP3 Proteases'. To investigate the versatile detection of DLPB protease activity sensing, four different peptide-DNA harpins were synthesized with peptide sequences designed for CTSB, MMP7, 3CL, and caspase-3 (see TABLE 1). The signal change was tested by incubating these DLPBs with their respective proteases. Each protease was incubated in its respective assay buffer, 3CL protease was incubated in commercial 3CL assay buffer (BPS Bioscience, Cat. No: 79956) while CTSB and MMP7 are incubated in 15 mM DTT in Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma Aldrich, Cat No: D8537), and CASP3 was incubated in PBS. Note that for detection of cell lysates, PBS is the buffer used as the proteases are assumed to be active in their native complex media. All DLPBs were incubated in 40 pL of their respective buffers and proteases.

[0195] CTSB DLPB LOD'. To determine the limit of detection for the CTSB DLPB, samples were tested with 30 nM, 20 nM, 15 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, InM, and 0 pM concentrations of CTSB. Each protease concentration was incubated with 1 nM CTSB DLPB in a final buffer composition of 15 mM DTT, 2 mM EDTA in IX DPBS. 10 pL of this solution was added to all required wells in a 96 black well plate. CRISPR buffer was then added to bring all volumes to 50 uL, quickly followed by 50 pL of RNP solution. To calculate the LOD the 3o/m method was used of a plot of fluorescence signal versus concentration was used to form a calibration curve.

[0196] CTSB DLPB Selectivity'. To determine the potential for off target signaling, the CTSB DLPB was treated with a variety of proteases to investigate any change in signal. For the assay a 350 pL solution of lOnM CTSB DLPB of 15mM DTT in DPBS was split into seven 40 pL aliquots. Into the tubes 3CL, MMP7. thrombin, CASP3, trypsin, and bovine serum albumin (BSA) were added individually to their respective aliquots to a final concentration of 20nM, along with a control where only buffer was added. These were then incubated for 30 minutes and read. These results were then compared to the signal when the CTSB DLPB is incubated with the same concentration of CTSB under identical conditions.

[0197] Comparison of Fluor ophore-Quencher 3CL DLPBs to Commercial Fluorogenic Substrates and CRISPR-Casl2a Amplification of DLPBs'. To evaluate the extent of the amplification of the CRISPR-Casl2a system provided on the DLPB signal, a comparison using a fluorophore-quencher (FQ) DLPB was used. As the FQ DLPB is cleaved via protease activity, the blocking and activating strand separate identically to regular DLPBs. Where the detection of FQ DLPBs differs from regular DLPBs is the inclusion of Cy5 fluorophore dye and a BHQ quencher on the DNA hairpins 5’ and 3’ ends. When in a hairpin, the close proximity of the dye to the quencher limits florescence, but after protease dependent melting of the hairpin structure, the dye is released, and its fluorescence can be measured. Due to this, the fluorescence signal from a FQ DLPB is 1 : 1 due to proteolytic cleavage, and therefore can be compared to both commercial fluorogenic substrates as well as DLPB signal generated from CRISPR-Casl2a amplification of DLPBs.

[0198] A FQ 3 CL specific DLPB was synthesized using the same DNA and peptide sequences as the CRISPR mediated DLPB, to give an accurate comparison to the CRISPR based system. It is noted that the addition of BHQ, and Cy5 modifications may have an adverse effect on the cleavage rate between FQ 3CL DLPBs and DLPBs, however the study attempted to mitigate this effect by designing these modifications to be as far from the peptide recognition site as possible on the probe. Therefore, this effect is believed to be mostly minimal due to the distance as well as the relative size of these modifications to the already present DNA in hindering the cleavage rates between the two DLPB substrates.

[0199] For assays including FQ 3CL DLPBs, 1000 nM of the FQ 3CL DLPB was incubated with 3CL protease in 3CL buffer for 30 minutes, then was diluted with CRISPR-Casl2a assay buffer to lOOnM in a black 96 well plate. The solution was then read using a plate reader. An excitation wavelength of 648 nm and an emission of 700 nm w ere used. It was noted that the Cy5 dye heavily and rapidly photobleached in this buffer, and therefore the first timepoint was taken as the signal for these assays. These results were compared to CRISPR mediated DLPB detection and commercial FQ 3CL probes at identical conditions. [0200] FQ 3CL DLPB Time-Dependent Assay Procedure'. Time dependent cleavage assays were also performed for the FQ 3CL DLPBs to evaluate the cleavage of 3CL DLPBs over time. Into a black 96 well plate, 100 nM of the FQ 3CL DLPB was added to 3CL buffer and 1 pM 3CL protease. The solution was then read using a BioTek Cytation 5 imaging multimode plate reader held at 25°C using an excitation wavelength of 648 nm and an emission of 700 nm . Controls containing only the RNP solution, and FQ 3CL DLPB without protease were also run. [0201] Cell Culture and Sample Preparation: The primary cell lines used in this study were NCI-H508 (CCL-253), RKO (CRL-2577), HT29 (HTB-38), and SW-620 (CCL-227). Every' primary⁷ cell line was purchased from the American Type Culture Collection (ATTC) and cultivated in RPMI-1640, fortified with 15% FBS, 1% penicillin/streptomycin, 1% Glutamax, 10% fetal bovine serum, 1% non-essential amino acids (sourced from Gibco), and lOmM HEPES, and kept in an environment-controlled incubator at 37°C with 5% CO2.

[0202] 2,000,000 viable cells were seeded into 6 cm tissue culture plates and left to incubate overnight. After incubation, the growth medium used to cultivate the cells was obtained to study the proteases secreted by the cells. For intracellular protease analysis, the cells were first rinsed with PBS (provided by Gibco). then physically dislodged via cell scraping the same PBS, and sonicated to lyse.

[0203] The cells were next collected into a pellet by centrifuging the solution at 16.2x g and a temperature of 4°C for a duration of 15 minutes. The supernatant was carefully collected for further analysis.

[0204] Detection of Protease Activity in Cell Lysates:

[0205] Detection of Protease in Activity in Cell Lysates with DLPBs'. To examine the activity of native proteases in complex media, lysates derived from colon cancer cell lines were subjected to treatment with DLPBs. Specifically, the primary cell lines NCI-H508, SW-620, RKO, HT29 were exposed to the CTSB, CASP3, MMP7, and 3CL DLPBs that are used in the study. In these cell samples, CTSB is expected to have high activity levels due to their observed higher expression and activity⁷ in previous studies [5-8], Conversely, protease activity for MMP7, CASP3, is not expected to be at higher activity than the CTSB. although their relative actives is not precisely known at the time of this study. The DLPB specific for the 3CL protease serves a general nonspecific control for non-specific/cross reactive cleavage in the system, as the presence of SARS CoV-2 proteases in these samples is not expected. Therefore, the activation of 3CL probes will serve as an estimate of the background in these systems.

[0206] To assess the functionality of detecting protease activity in the cell lysates samples, 10 nM of all DLPBs were incubated with 15 pL of the cell lysates (~2 million cells/mL) in a total volume of 45 pL of IX PBS. The samples were then incubated for 30 minutes to allow for DLPB cleavage. After incubation, 10 pL aliquots of the samples were then diluted to 50 pL with CRISPR buffer and then added to RNP for a final concentration of 1 nM DLPB, lOnM RNP, in 90%: 10% CRISPR buffer PBS mixture. The RNP control also was created with this buffer composition and ran alongside the DLPBs. Samples were read in a CRISPR assay.

[0207] Detection of 3CL Protease in 3CL Spiked Cell Lysates Using 3CL DLPBs. To determine the ability of DLPBs to detect a specific protease of interest in complex media. NCI- 14508 lysates were spiked with 3CL protease and treated with 3CL DLPBs. Any enhancement in the signal over a control of 3CL DLPBs treated with the cell line w ould indicate the ability of the DLPB to function in complex media. 10 nM of the 3CL DLPB was incubated with 15 pL of the NCI-H508 cellular lysates with and without 1 pM 3CL protease for 30 minutes in 3CL buffer. The mixture was then added to RNP for detection, along with controls.

[0208] Detection of CTSB Protease in Cell Lysates with Commercial CTSB Reporter'. To validate that the signal obtained from incubating DLPBs comes from CTSB activity, a commercial CTSB reporter was also treated with the cellular lysates to support the presence of CTSB in the samples. The commercial probe was incubated at the same concentration as the DLPBs, 10 nM, as well as the manufacturer-recommended concentration of 1 pM. Both concentrations were incubated with 3 pL of SW-620 cell lysates in 40 pL PBS, shaking at 1500 RPM for 30 minutes. After incubation, 10 pL of each sample w as added to 90 pL of CRISPR buffer and read at Ex: 380 nm Em: 460 nm.

[0209] LOD of Cellular Lysates with DLPBs'. To test the detection threshold using DLPBs on cell lysates, the CTSB DLPB was treated with varying concentrations of the most responsive cell line, SW-620. lOnM of the CTSB DLPB was incubated with 15 pL, 5 pL, 3 pL, and lower volumes of cell lysates and incubated in lx PBS for 30 minutes. Volumes for cell lysates lower than 3 pL were omitted due to a lack of substantial signal over the background.

[0210] CTSB DLPB Detection of Cellular Lysates with Inhibitor'. To validate that the response from incubating DLPBs with cellular lysates is due to specifically CTSB activity, the detection of protease activity in cell lysates with DLPBs was repeated, this time including a commercial CTSB inhibitor (Sigma Aldrich, CTSB Inhibitor II, Ac-LVK-CHO Cat. No. 219385- IMG). lOnM of the CTSB DLPB was incubated with 3 pL of SW-620 cell lysates in lx PBS with 30 pL of 1 rnM concentration CTSB inhibitor.

[0211] Commercial CTSB Probe Detection of Cellular Lysates with Inhibitor'. A commercial CTSB fluorogenic probe was incubated with SW-620 cellular lysates in the presence of a CTSB inhibitor to support the signal increase was due to active CTSB. 10 nM and 1000 pM of the commercial probe were incubated with 3 pL of SW-620 cell lysates and 30 pL of CTSB inhibitor in PBS. After a 30-minute incubation, 10 pL of each sample was added to 90 pL of CRISPR buffer and read at Ex: 380 nm Em: 460 nm.

[0212] Gel Electrophoresis Results:

[0213] PAGE Gel Characterizations of DNA, Peptide-DNA Conjugates, Linker -Peptide- DNAs, and DLPBs. Polyacrylamide gel electrophoresis (PAGE) was utilized to reinforce the MALDI-MS characterization of DNA, peptide-DNA conjugates, linker-peptide-DNAs, and DLPBs. The 15% acrylamide gels, which were produced in-house, were made by diluting a stock 40% acrylamide solution (Bio-Rad, 40% Acrylamide/Bis Solution 19:1, Cat no: 1610144) with IX Tris-Borate-EDTA buffer (TBE). The IX TBE was produced in-house by diluting 10X TBE (Fisher BioReagents. Tris-Borate-EDTA 10X. Cat No: BP1333-4) to 15%. This was followed by the addition of 50 pL N.N.N'.N'-Tetramethyl-ethylenediamine (Sigma Aldrich, N,N,N’,N’-Tetramethyl-ethylenediamine, Cat No: T9281), and finished by adding approximately 2 mg of ammonium persulfate (Fisher BioReagents, Ammonium Persulfate (APS) Electrophoresis. Cat no: BP17). After mixing well, this solution was poured into a Mini- PROTEAN Tetra Handcast System (Bio-Rad. Cat. No: 1658050). A ten-well comb was placed at the top of the gel, which was allowed to solidify for about two minutes. Following the removal of the comb, the gel wells were thoroughly rinsed with water. The gel was then installed in a Mini-PROTEAN Tetra System (Bio-Rad, Cat No: 1658005EDU) and filled to the manufacturer's recommended line with IX TBE buffer.

[0214] For PAGE characterization, 15 pL volumes of all samples were prepared, all having an approximate final concentration of IpM (DNA, peptide-DNA, linker DNA, and DLPBs). Samples were mixed with 2 pL of glycerol and diluted to 15 pL with TBE buffer. A nucleic acid ladder was prepared, which included 0.2 pL of an ultralow DNA molecular weight ladder (Invitrogen, Tracklt Ultra Low Range DNA Ladder, Cat No: 10488023), 2 pL of glycerol, and was likewise diluted to 15 pL with TBE. 10 pL of all samples were loaded into different wells of the prepared 15% PAGE gel. To track gel progression, 10 pL of a gel loading dye (Sigma Aldrich, Gel Loading Solution, Cat No: G7654) was added into a dedicated well.

[0215] The gel was run at a voltage of 120V until the loading dye had traveled -90% of the gel's length, at which point it was stopped. The gel was then removed from the holder, placed in approximately 50 mL of water, and stained with 2 pL of GelRed nucleic acid stain (Milipore Sigma, GelRed Nucleic Acid Stain (10,000X, Water), Cat No: SCT123) for 10 minutes. Finally, the gel was placed into a Bio-Rad ChemiDoc MP imaging system (Bio-Rad, Cat No: 12003154), and the GelRed stain was monitored at Ex: 590 Em: 110. [0216] PAGE Gel Purification ofFQ 3CL DLPBs\ In addition to HPLC purification, PAGE gel purification was also attempted on the DLPBs to test the versatility of purification. For gel purification, a 15% PAGE gel was created using an 8 M urea (Fisher Chemical, Urea, Cat No: U15-500) solution in water in place of TBE buffer in order to create a denaturing gel. Into the gel, 20 nmol of the FQ 3CL DLPB was added run. The expected species in this gel are peptide- DNA quenchers, dyed blocking strands, and the final FQ 3CL DLPB structure. Since each of these species have a dye that can be seen by eye. and it was observed that there were numerous well separated bands. The topmost band (expected FQ 3CL DLPB due to migrating the least dow n the band or by having the highest observed m/z) was extracted by use of a razorblade, placed into a 15 mL tube. The gel was then crushed using a pipette tip and 3 mL of DI water was added to the tube. The mixture was then frozen using liquid nitrogen and thawed three times to aid in the crushing of the gel, then placed on a tube rotator for 48 hours. At this point it was noted that the blue color has migrated from the gel fragments into the solution, indicating that the present species had migrated into the DI water. The gel fragments were separated via centrifugation and passage through a fritted syringe, and the resultant solution was washed 10 times using a 3K spin filter to remove excess urea. The solution was then characterized via UV-Vis and the presence of purified FQ 3CL DLPBs were confirmed via MALDI. The approximate yield of this separation was 30%, exceeding what was achieved via HPLC. However, this compound was observed to slightly more impure than HPLC purified DLPBs though MALDI-MS and the melting assays.

[0217] UV-VTS Data:

[0218] DBCO Azide Click Chemistry. UV-Vis spectroscopy (Agilent, Cary' 60 UV-Vis) was used to characterize DNA concentrations, presence of DNA modifications (DBCO, dye, quencher, etc.,) as well as completion of copper-free click chemistry.

[0219] Melting Temperature Analysis:

[0220] Melting Assays of DLPBs and FQ 3CL DLPBs: To investigate the hypothesized DLPB conformations of the DLPBs, the study conducted commonly performed melting assays. These assays involve heating DNA that possesses secondary structures; the process increases the temperature to a point where it disrupts the hydrogen bonds in double-stranded DNA. leading to its conversion into single-stranded DNA. Although changes in DNA structure can be detected through absorbance methods, the study opted for fluorescence-based methods utilizing intercalating dyes and fluorophore-quencher systems, due to their superior signal intensity. Consequently, when melting assays reveal a change in fluorescence, it suggests the presence of a secondary structure within the system. By measuring the temperature at which this change occurs and comparing it to the anticipated melting temperature for DLPB structures, the study can corroborate the existence of the desired DLPB conformations.

[0221] Melting Assays of Purchased DNA Hairpins'. To evaluate the capability of confirming hairpin and non-hairpin structures through melting assays, commercially synthesized DNA hairpining and non-hairpining sequences were purchased as references. Specially, from TABLE 3, DNA Hairpin 12-8 and DNA Non-Hairpin 12-0 (where 12 indicates the number of extra T-bases in the corresponding hairpin loop to simulate peptide and linker length, and 8 and 0 indicate the number of complementarity bases in the DNA stem) were used to simulate DLPBs in their stem and loop size, while their overall length and activating sequence was held constant. To distinguish between single-stranded and double-stranded DNA, an intercalating dye, SYBR Green (Millipore Sigma, SYBR Green Nucleic Acid Gel Stain, S9430), was added to the DNA solutions at a final concentration of 2X. This dye specifically binds to double-stranded DNA, producing a strong signal, whereas it does not bind to single-stranded DNA, resulting in diminished signal. Thus, a decrease in signal intensity with this dye signifies the transition from double-stranded to single-stranded DNA. For the melting temperature assay, 10 pL of 1 pM DNA samples mixed with 2X SYBR Green in solutions containing IX CRISPR buffer and buffer were used. These mixtures were added to 96 well plate (Bio-Rad, Hard-Shell High Profile 96-Well Semi-Skirted PCR Plates, Cat. No: HSS9601) and the fluorescence was measured. Control samples including 2X SYBR Green, IX CRISPR buffer, and 50 mM NaCl were also prepared and analyzed using a qPCR instrument (Bio-Rad, CFX96 Real-Time System C l 000 Touch Thermal Cycler), specifically monitoring the SYBR Green fluorescence channel. All solutions were prepared and measured in triplicate. A run following the same procedure as the one listed above w as also completed in 50 mM NaCl instead of CRISPR buffer.

[0222] The specific qPCR method used was detailed as follows: Throughout the assay, the lid temperature was maintained at 105°C. The samples were gradually heated to 90°C over a span of 5 minutes, after which they were maintained at this temperature for 30 seconds. Subsequently, the samples were cooled to 4°C, at which point the initial reading was taken in the selected fluorescence channel. The temperature was then increased incrementally by 0.5°C. Readings were taken each time the temperature reached the specified increment in channel of choice. For this assay the SYBR green channel was read. This process continued, with temperature increments and readings, until reaching 95°C.

[0223] qPCR Melting Assays of FQ 3CL DLPBs. To verify the hypothesized DLPB structure of the DLPBs, melting assays were conducted using DLPBs labeled with fluorophores and quenchers (FQ). In their hybridized state, the proximity of the fluorophore to the quencher suppresses the fluorescence signal. Dehybridization increases the distance between these molecules, leading to the emission of a fluorescence signal. This contrasts with the use of intercalating dyes, where increases in fluorescence indicate the transition from double-stranded DNA to single-stranded DNA.

[0224] The procedure for the melting assays of FQ-labeled DLPBs was as follows: 10 pL of 1 pM DLPBs. along with DNA quencher strand, Cy5-labeled blocking strand, and a control mixture of peptide-DNA quencher and non-conjugated dye-labeled blocking strand, were prepared in both IX CRISPR buffer. These were also compared against controls of IX CRISPR buffer. Samples were placed into a 96 well plate (Bio-Rad, Hard-Shell High Profile 96-Well Semi-Skirted PCR Plates, Cat. No: HSS9601) and analyzed using the qPCR machine, specifically monitoring the Cy5 fluorescence channel. To account for photobleaching of the Cy5 dye, the change in fluorescence for the Cy5-labeled blocking strand was normalized to one, and this correction factor was applied to other samples. No change in signal from the DNA quencher strand was observed, leading to its exclusion from further analysis. The purpose of the control mixture was to benchmark the melting temperature of the intermolecular hybridization against the expected intramolecular DLPB structure of the DLPBs. A significant increase in the melting temperature would suggest successful DLPB structure conjugation. A run following the same procedure as the one listed above was also completed in 50 mM NaCl instead of CRISPR buffer.

[0225] The qPCR method monitored in the Cy5 channel.

[0226] qPCRMelting Assays ofFQ 3CL DLPBs Incubated with 3CL Protease'. To support the hypothesis that proteases can mediate a cleavage in the peptide region which causes a conformational change decreasing the melting temperature of the probes, the FQ 3CL DLPB was treated with 3CL protease and underwent melting temperature analysis. Into a 1.5 mL centrifuge tube, 1 pM of the FQ 3CL DLPB was incubated with 3 pM of 3CL protease overnight. It was estimated from the time dependent cleavage of the FQ 3CL DLPB that 1 pM of 3CL protease is able to cleave -100 nM of FQ 3CL DLPB substrate in 2 hours, therefore after 8 hours approximately all of the FQ 3CL DLPB should be cleaved and show a considerable melting temperature shift. After the FQ 3CL DLPB was incubated with 3CL protease at the above concentrations in 3CL buffer, 10 pL of the solution was then split into 10 pL aliquots in a 96 well plate (Bio-Rad, Hard-Shell High Profile 96-Well Semi-Skirted PCR Plates, Cat. No: HSS9601) and analyzed using a qPCR monitoring the Cy5 channel. A control containing just the signal of the cyanine 5 dye strand in 3CL buffer was also run, and from this response a correction factor was created to account for photobleaching which was used in the data analysis of FQ 3CL DLPB species. Controls of 3CL buffer, BHQ stand, and luM 3CL protease were also run, but omitted due to a lack of considerable signal.

[0227] Data Analysis:

[0228] Definitions for Relative Fluorescence and Relative Signal'. Relative Florescence is defined by the change in fluorescence values of a probe treated with protease in comparison to the fluorescence values of the RNP complex, or to that of the probe without treatment from protease. In the manuscript, if the relative fluoresce is not defined, it is assumed to be relative to the probe minus protease. In case where it is relative to the RNP, these are clearly defined.

[0229] Relative fluorescence was calculated using Equations 1 and 2:

Id

Relative fluorescence = __{Protease t} eq (1) probe,t

Relative fluorescence_RNP = - — le ’■ —t eq (2) IRNPU

[0230] Relative Signal is defined by the change in fluorescence values from a probe treated with protease in comparison to a probe without protease treatment, each with a subtraction of the background fluorescence values of the RNP complex.

[0231] Relative signal was calculated using Equation 3: where IpPf^^ease denotes fluorescence values of a probe treated with protease, lfrobe^ease without protease, and 1_RNP as the RNP fluorescence values.

[0232] Definitions for Reported Error and Propagation of Error'. The reported error for non-derived raw data values, such as fluorescence results (RFU), is expressed as the standard deviation of at least three replicate measurements, unless otherwise noted. For derived results, like enhancement factors (EF), errors are calculated by determining the percent standard deviation of the DLPB plus protease signal at a specific time point, and this percent error is then applied to the calculated result to find absolute errors for derived measurements. This approach is adopted because the majority of the uncertainty in these measurements primarily comes from the variability in the DLPB plus protease signal, rather than from signals related to the RNP complex or DLPB minus protease. Traditional error propagation rules are not utilized in this scenario, as they inadequately account for the true variation and disproportionately affect the calculated result.

Additional Results and Discussion

[0233] TABLE 4-8 show melting temperature analysis.

TABLE 4. Melting temperature analysis of DNA hairpins and dsDNA at various DNA concentrations. Values represent expected melting temperatures of the species in degrees Celsius. Calculations were done using mFold software at a constant 40 mM Mg²⁺, 100 mM Na⁺, and a loop size of ten nucleotides.

TABLE 5. Melting temperature analysis of DNA hairpins and dsDNA at various Na⁺ concentrations. Values represent expected melting temperatures of the species in degrees Celsius. Calculations were done using mFold software at a constant 40 mM Mg²⁺, InM DNA. and a loop size of ten nucleotides.

TABLE 6. Melting temperature analysis of DNA hairpins and dsDNA at various Mg²⁺ concentrations. Values represent expected melting temperatures of the species in degrees Celsius. Calculations done using mFold software at a constant 100 mM Na⁺, InM DNA, and a loop size of ten nucleotides.

TABLE 7. Melting temperature analysis of DNA hairpins and dsDNA at various loop lengths. Values represent expected melting temperatures of the species in degrees Celsius. Calculations done using mFold software at a constant 100 mM Na⁺, InM DNA, and 20 mM Mg²⁺.

TABLE 8. Melting temperature analysis of DNA hairpins and dsDNA at various loop lengths. Values represent expected melting temperatures of the species in degrees Celsius. Calculations were done using mF old software at a constant 100 mM Na⁺, InM DNA, and 40 mM Mg²⁺.

[0234] TABLES 9-10 show MALDI-MS data of 3CL, CASP3, CTSB, and MMP7 DLPBs and intermediates and MALDI-MS data of fluorophore quencher DLPB.

TABLE 9. Expected (Ex) Versus Actual (Ac) MALDI m/z of all DLPB and Intermediate

Synthetic Species

TABLE 10. Expected (Ex) Versus Actual (Ac) MALDI m/z of FQ 3CL DLPB.

[0235] UV-Vis Data:

[0236] Peptide-DNA Conjugation via DBCO Azide Click Chemistry. Activator DNA was treated with increasing concentrations of azide pep-3CL for 24 hours and the reaction progression was observed though monitoring the DBCO’s 310nm absorbance. Treatment of 0: 1, 1: 1, 3: 1, and 5: 1, azide to DBCO was measured. In samples of 3: 1 and 5:1 equivalents the 310 nm peak appears to have disappeared indicating a successful full conjugation. Therefore, all DBCO azide click chemistry used at least a 3: 1 ratio to ensure complete product conversion. [0237] Peptide-DNA Conjugation via DBCO Azide Click Chemistry. UV-Vis spectroscopy was used to confirm attachment of 8-nt blocking DNA to peptide-DNA linkers. 5: 1 molar ratio was used with respect to DBCO 8-nt blocking strand to peptide-DNA linkers. Therefore, with 100% conjugation a 20% absorbance decrease at 310 nm is theoretically expected. Upon treatment the DBCO’s characteristic absorbance at 310 nm decreases 18%. indicating that the azide on the peptide-DNA-linker has successfully conjugated.

[0238] UV-Vis Spectra ofFQ 3CL DLPB Probe'. UV-Vis spectroscopy was used to support successful synthesis of FQ 3CL DLPB Probes. A UV-Vis spectrum confirms the presence of DNA, BHQ, and cyanine-5 due to their characteristic absorbances at 260 nm, -580 nm, -650 nm respectively. The absence of DBCO’s characteristic 310 nm peak indicates successful conjugation of quencher-DNA linker species and dye labeled blocking strands.

[0239] FQ 3CL DLPB and Commercially Synthesized DNA Hairpin Melting Assays: [0240] Commercially Synthesized DNA Hairpin Melting Assays: To test the ability to support DLPB structures via melting assay, commercially purchased DNA Hairpin 12-8 and the control Non-hairpin 12-0 sequence underwent melting temperature analysis. Upon heating both species in both 50 mM NaCl and CRISPR buffer, a distinct absorbance change is observed in the commercial 12-8 DLPB at much higher temperatures than the Non-hairpin 12-0 sequence. The expected melting temperature in CRISPR buffer is 52°C for Hairpin 12-8 and 16.2°C for DNA Non-hairpin 12-0 when at 1 pM. The expected melting temperature in 50 mM NaCl is 41°C for Hairpin 12-8 and 8.4°C for DNA Non-hairpin 12-0 when at 1 pM. Which match almost as expected to the data, save for the NaCl melting which is slightly higher than expected. However relative differences between the hairpin and non-hairpin sequences are the best indicator of which supports that hairpin/non-hairpin structures can be analyzed by this method. These results are shown in FIGS. 8A-8B.

[0241] FQ 3CL DLPB Melting Assays : In order to further support the hairpin conformation of DLPBs melting assay s were completed on FQ 3CL DLPBs. As the FQ probes are heated, dehybridization of the stem causes the quencher and fluorophore to separate, increasing fluorescence signal.

[0242] FQ 3CL DLPB Melting of Impure and Purified Species: FQ 3 CL DLPB was collected before and after gel purification and was subjected to melting temperature analysis in 50 mM NaCl. As a control, the dye-labeled blocking DNA (Cy5 strand) and quencher-labeled activating DNA (BHQ strand) were also subjected to melting temperature analysis individually, as well as a 1 pM mixture of the species. The signal of the Cy5 strand was used to correct for photobleaching and temperature dependent fluorescence. The signal for 50 mM NaCl, and BHQ strand was omited due to lack of considerable signal. The inclusion of the mixture of Cy5 and BHQ serves as an unconjugated, i.e. non hairpin DNA hybridization, control. Therefore, the melting of this mixture will serve as the baseline in comparison to the FQ 3CL DLPB, wherein if the melting temperature of the DLPB is higher than this control, it will indicate successful conjugation of the DLPB into a hairpin structure. Upon heating the samples, Cy5 and BHQ strands, impure 3CL DLPB, and pure 3CL DLPB, clear differences in the fluorescence response are observed. The expected melting temperature in 50 mM NaCl is 12.2°C for the Cy5 BHQ mixture and 41 °C for the DLPB when at 1 pM. Between the Cy5 and BHQ strand control and the FQ 3CL DLPB, an increase in the melting temperature is observed ~20°C. This indicates that the FQ 3CL DLPB structure does form a hairpin like structure. The observed melting temperature for the species seems to be ~20°C higher than expected. [0243] It is also observed that the impure FQ 3CL DLPB appears to be a convolution of the mixture control and pure FQ 3CL DLPB, indicating that there are unconjugated impurities present. After purification, this melting pattern is not observed, indicating that the purification was successful in removing non-hairpin impurities. These results are shown in FIG. 8C.

[0244] FQ 3CL DLPB Melting Assay in CRIPSR Buffer'. FQ 3CL DLPB was subjected to melting temperature analysis in CRISPR buffer to support the hypothesized structures in the assay buffer. As a control, the dye-labeled blocking DNA (Cy5 strand) and quencher-labeled activating DNA (BHQ strand) were also subjected to melting temperature analysis individually, as well as a 1 pM mixture of the species. The signal of the Cy5 strand was used to correct photobleaching and temperature dependent fluorescence. Upon heating the FQ 3CL DLPB in the assay, a melting temperature increase is seen in the DLPB over the control mixture. This indicates that the DLPB is forming a hairpin structure in the assay buffer and interacting with the Cast 2a enzymes in the predicted manner. It is noted that the fluorescence of Cy5 is considerably quenched in the CRISPR buffer relative to 50 mM NaCl, and there seems to be a larger photobleaching effect at higher temperatures as seen in the Cy5 and BHQ mixture, which even after correcting with the Cy5 photobleaching is still present. However, this decease does not affect overall melting temperature analysis of the species. The expected melting temperature in CRISPR buffer is 27°C for the Cy5 BHQ mixture and 52.2°C for the DLPB when at 1 pM. The DLPB in this system has a melting temperature of ~ 64°C, much higher than what is theoretically calculated, possibly indicating that the peptide plays a role in stabilizing the hairpin conformation. These results are shown in FIG. 8D.

[0245] Gel Electrophoresis Results: FIG. 9A shows Gel Electrophoresis of Activator DNA, Peptide-DNA Conjugates, Linker-Peptide-DNAs, and DLPBs. FIG. 9B shows PAGE Separation of FQ 3CL DLPBs

[0246] Time Dependent Treatment of FQ 3CL DLPBs with 3CL Protease: Upon incubation of FQ 3CL DLPBs with 3CL protease, the cleavage of the peptide region will similarly cause DNA dehybridization of the DLPB to the CRISPR mediated detection platform. However, disassociation in the FQ 3CL DLPB will cause a direct enhancement of florescence due to the dissociation of the Cy5 dye and BHQ. This probe serves as a control to further support the intended DNA disassociation of DLPBs and will serve as a baseline to compare to the CRISPR systems to gauge the effect of CRISPR amplification on the system. 100 nM of the 3CL FQ DLPB was incubated with 1 pM 3CL protease and the florescence measured. These results are shown in FIG. 10.

[0247] CRISPR-Casl2a Assays: [0248] Effect of Activating DNA Concentration on CRISPR- Cas 12a Assays: To evaluate which concentration of DNA is most optimal for CRISPR-Casl2a assays, RNP was held at a constant 10 nM was treated with increasing concentrations of a ssDNA activator. It was observed that an increased concentration of activator DNA correlates to an increased CRISPR- Casl2a fluorescent response. These results are shown in FIG. 11A.

[0249] Effect of Increasing RNP Concentration: To discover an optimal RNP concentration for CRISPR assays, fixed InM of CRISPR-Casl2a activator DNA was treated with RNP concentrations of 100 nM, 10 nM, 1 nM and 0.1 nM to observe signal enhancement versus time. Into a black 96 well plate 49 pL of CRISPR-Casl2a assay buffer, and IpL of lOOnM CRISPR-Casl2a activator DNA, and 50 pL of two times concentrated RNP solutions were directly added to a final volume of 100 uL. These samples were then subjected to monitoring. Concentrations of RNP below InM show incomplete saturation within 60 minutes while concentrations around lOnM saturate within the time frame. An RNP concentration of lOnM was chosen due to its ability to saturate within one hour with 1 nM of DNA activator. These results are shown in FIG. 11B.

[0250] Effect on DNA Activators with Protease Incubation in CRISPR-Cas 12a Signaling: To investigate the effect of protease incubation on DNA only CRIPSR assays, 10 nM of DNA activator was incubated with 3 pM 3CL protease for 2 hours and then added to a CRISPR assay. In doing so there was no change seen with the activation of the DNA activator with and without protease incubation. This implies that certain proteases have no effect on the ability of CRISPR Casl 2a signaling or that they are able to interfere with the binding of activator DNA with RNP complexes. These results are shown in FIG. 11C.

[0251] CRISPR Activation with Activator DNA Hybridized to 24-nt and 16-nt Blocking DNA: To test the activation of hybridized dsDNA on the activation of CRISPR-Casl2a, 1 nM ssDNA activator strand was treated with 10 nM of the 16-nt blocking as well as the 24-nt blocking DNA. These were mixed immediately and added to the CRISPR procedure. The Activator DNA appears to be significantly restricted when the full complement is added yet shows no restriction with the partial complement. This suggests that CRISPR cannot access fully double-stranded sequences, while, with partial complements. CRISPR-Cas 12a can still access the activating sequence. Though DLPBs do not exhibit complete blocking, it is hypothesized that the DLPB's loop structure might achieve a similar restriction in activating CRISPR-Casl2a prior to cleavage. Based on these observations, it is further hypothesized that DNA DLPBs might also be inhibited from activating CRISPR unless converted to ssDNA. These results are shown in FIGS. 11D-11E. [0252] Hypothesized Activation of CRISPR-Casl2a with DNA Activator, DNA Activator and 16-nt Blocking DNA, and 24-nt Blocking DNA'. Treatment of CRISPR-Casl2a enzymes with DNA activators and blocking DNAs was accomplished to see to what extent blocking would have on the activation of the CRISPR-Casl2a system. It was observ ed that activator DNA with and without treatment of the 16-nt blocking DNA was able to activate CRISPR- Casl2a with no impediment. However, when Activator DNA was treated with the 24-nt blocking DNA heavy restriction on CRISPR-Casl2a activation was observed. It is hypothesized that the unblocked potion remaining from C16 binding can cause toehold mediated displacement, causing activation even in partially blocked systems. This hypothesized activation is shown in FIGS. 12A-12C.

[0253] CRISPR Activation with Large Loop DNA Hairpins and Blocking DNAs'. 1 nM activating 30-8 DNA hairpins were tested with 10 nM of a partial 16-nt blocking DNA as well as a 24-nt blocking DNA to see restriction on CRISPR signal. Hairpin and non-hairpin activators were added immediately to lOnM of the blocking DNAs and subjected to the CRISPR-Casl2a procedure. It was observed that 24-nt blocking DNAs again fully disallow the activation of CRISPR-Casl2a enzymes. However, with treatment of the 16-nt blocking DNA. an initial inhibition is seen followed by an activation with looks similar to a positive cooperativity curve, indicating that the enzyme is able to free substrate to activate more CRISPR-Casl2a enzymes. This trend may be attributable to CRISPR-CAS12A cleaving the DNA hairpin structure, leading to the release of single-stranded activating DNA. This specific issue may not arise in DLPBs since their loop structure comprises a peptide, rendering them resistant to cleavage by CRISPR-CAS12A enzymes. These results are shown in FIGS. 13A- 13B

[0254] CRISPR-Casl2a Studies with Blocked DLPBs. To test the interaction of protease incubated DLPBs, and DNA activators, with blocking DNA, lOnM of the 3CL DLPB and DNA activator w ere treated with 100 nM of the 16-nt blocking DNA and incubated with 4 pM 3CL protease for 2.5 hours. Samples were then taken and subjected to the CRISPR-CAS12A procedure. Upon treatment with protease, DLPBs demonstrated an increase in relative signal; however, samples containing the blocking strand exhibited minimal variance in activation, with or without the strand. Based on these findings, all subsequent assays were conducted without any blocking DNA, suggesting that DLPBs do not require complete blocking of their activation sequence to prevent background activation. These results are shown in FIG. 13C. It should be noted that these assays were performed using unpurified DLPBs, and further analysis revealed that background activation was primarily due to activating impurities rather than nonspecific hairpin cleavage.

[0255] Hypothesized Interaction Between DNA Hairpins and CRISPR-Casl2a Enzymes'. Experimental results indicated that CRISPR-Casl2 was able to activate using 30-8 DNA hairpins. This activation can come from a few possible sources including the presence of nonhairpining impurities in the sample, slow disruption of the hairpin stem by CRIPSR-Casl2a enzymes, or the minor presence of non-hairpining DNA in solution. Each of these possibilities can start the activation of the CRISPR-Casl2a enzymes. After this initial activation, CRISPR- Casl2a enzymes having the ability to cleave ssDNA, can cleave the loop region of hairpins causing them to degrade into new activating DNA causing an amplification cycle. This hypothesized activation is shown in FIGS. 14A-14B.

[0256] Hypothesized Further Interactions Between DLPBs and CRISPR-Casl2a Enzymes'. Experimental results indicated that DLPBs do not exhibit the same background activation as DNA hairpins. It is hypothesized that this is the case because active CRISPR-Casl2a enzymes cannot cleave DLPBs to generate activating DNA, contrary to what is hypothesized in DNA hairpin systems. Active CRISPR-Casl2a can indiscriminately cleave ssDNA into fragments; however, with DLPBs, the only section of exposed ssDNA is within the activating sequence. Therefore, any cleavage within the loop of the DLPB structure will result in the generation of inactive DNA fragments, without the possibility of further activating CRISPR-Casl2a enzymes. This hypothesized interaction is shown in FIG. 15.

[0257] Effect of Purification on DLPB CRISPR-Cas 12a Response'. Impure and HPLC purified samples of 1 nM 3CL DLPBs were incubated with 3 pM 3CL protease for 2 hours and then subjected to the CRISPR protocol. Impure DLPBs show high background, likely due to the presence of non-hairpining unreacted species, while after HPLC purification, DLPB mixtures show retention of the same signal seen in impure samples but with the decrease of signal in unincubated samples. These results are shown in FIGS. 16A-16B.

[0258] Effect of DLPB Concentration on CRISPR- Cas 12a Assays: 10-wise dilutions of 3CL DLPBs from 10 nM to 10 pM were incubated with 3 pM of 3CL protease for 2 hours and then subjected to a CRISPR-CAS 12A fluorescence assay. These results are shown in FIGS.

16C-16F

[0259] Effect of Protease Incubation Temperature on DLPB CRISPR- Casl2a Assays: CRISPR-Casl2a assays were run at both 25°C and 37°C temperatures to evaluate what extent the cleavage rate of proteases and stability of the DLPB have dependent on increased temperature. Incubation of 10 nM DLPB was held at 25°C and 37°C with 2 pM 3CL protease for 2 hours and the resulting solution was read at 25°C. Heating was shown to increase the background signal of DLPBs without protease treatment while there is no discernable difference in signal between DLPBs incubated with protease. Subsequent experiments were run at 25°C. These results are shown in FIGS. 16G-16H.

[0260] Limit of Detection Studies of 3CL DLPBs: Half-wise dilutions of stock 3CL protease were created in 3CL assay buffer and added to 10 nM aliquots of the 3CL DLPB. Samples were then incubated for 30 minutes and read using the CRISPR-Casl2a assay procedure. To calculate the LOD the 3o/m method was used of a plot of fluorescence signal versus concentration was used to form a calibration curve. These results are shown in FIGS. 16I-16J. [0261] One-pot Assay Detection of 3CL Protease: The modified one-pot detection procedure was carried out using 1 nM 3CL DLPB to detect 500 nM and 1000 nM concentrations 3CL protease. The samples were incubated with protease for 30 minutes and then read with the modified procedure. Increases in response after protease incubation were observed with both protease concentrations, and was similar to the two-pot procedure, supporting the viability of the simplified detection protocol. These results are shown in FIG. 16K

[0262] Comparison of Commercial Protease 3CL Fluorogenic Substrate Probes to CRISPR Mediated 3CL DLPBs and FQ 3CL DLPBs: To confirm the presence of protease activity and support/benchmark the ability of DLPBs to detect protease activity, commercial 3CL protease reporters were employed in this study. The commercial substrates were both used to confirm the purchased proteases are active, but also to see the detection differences between what is commercially available and the DLPB systems. Note that the commercial substrates were used at both 100 nM, which is an average concentration advised by the manufacturers, and 1 nM as the same as the DLPBs are kept in assays.

[0263] Comparison of 3CL Protease Detection with Commercial 3CL Fluorogenic Substrate, FQ 3CL DLPBs, and CRISPR Mediated 3CL DLPBs: To test the differences between normal peptide substrates to the modified DNA-Peptide-DNA triblock system, a comparison between commercial 3CL fluorogenic substrates and a FQ 3CL DLPB was done. The sequence between the fluorogenic substrate and the FQ 3CL DLPB is held to be near constant, so this comparison aims to tell the difference between cleavage rates between the normal substrate and the DNA modified substrate. It was hypothesized that the addition of DNA w ould sterically hinder the protease from cleaving the unnatural substrate, and therefore the overall response would decrease. To investigate if this were the case, 100 nM of both the commercial 3CL fluorogenic substrate and 100 nM of the FQ 3CL DLPB were incubated with 3CL protease for 30 minutes and read. It was observ ed that there was a significant drop in the response of the FQ 3CL DLPB relative to the non-DNA modified fluorogenic substrate with the response gain (at 250 nM 3CL protease) at 1.55 to 2.87-fold respectively. However, this initial decrease may be outweighed by the amplification that the DNA handle provides via CRISPR-Casl2a signaling.

[0264] Therefore, to test the capabilities of the DLPB to the commercial substrate, an assay comparing the signal response at the same protease concentrations (250 nM and 500 nM) was also accomplished. The commercial substrate was incubated again for 30 minutes at 1 nM and 100 nM final substrate concentrations. It was observed that at 1 nM of the 3CL commercial substrate shows no increase in signal, and therefore the study compared the commercial substrate at a concentration of 100 nM to the DLPB system at 1 nM. Use of 100 nM DLPB in this case would be cumbersome on the reagents required and therefore not used as a comparison. 1 nM of the 3CL DLPB was incubated with 250 nM and 500 nM 3CL protease for 30 minutes and introduced to RNP to be detected. Upon comparing 100 nM of the commercial 3CL substrate to 1 nM of the 3CL DLPB, it was noted that at even 100-fold less concentration, the DLPB shows a 2.5-fold increase over the commercial substrate. These results are shown in FIG. 17.

[0265] Additional Cell Study Results:

[0266] Delection of 3CL Protease in 3CL Protease Spiked Cell Lysates'. To determine the ability of DLPBs to detect specific proteases in complex media, the 3CL DLPB was treated with the NCI-H508 cellular lysates. Incubation in complex media was carried out by using 15 pL of the NCI-H508 cell line in a total volume of 45 uL of 3CL buffer. Upon incubation of the DLPB with the cell lysates an enhancement vs RNP of approx. 4-fold is seen. However, the enhancement vs RNP of the sample incubated with cell lysates and 1 pM 3CL protease was approx. 13-fold. This indicates that the probe does work as intended in complex media and is able to detect specific proteases in such conditions. These results are shown in FIG. 18A.

[0267] Cell Line Response From CTSB, 3CL, MMP 7, and CASP3 DLPBs : All DLPBs were incubated with NCI-H508, HT29, RKO and SW-620 cell lysates. DLPBs were incubated for 30 minutes with 15 pL of all mentioned cell line lysates and read at a final concentration of 1 nM and read via a plate reader. It was observed that the signal generated from the CTSB DLPB was increased relative to the all other DLPBs, with their response all ~2-fold. These results are shown in FIGS. 18B-18E.

[0268] Time Dependent LOD of SW-620 Lysates'. CTSB DLPBs were incubated with volumes of the SW-620 cell lysates (~2 million cells / mL) to determine a crude LOD for colon cancer cells. CTSB DLPBs were incubated with SW-620 cell lysates and the freed DNA was detected using CRISPR Cast 2a. It was observed that volumes below 3 pL of cell lysates did not return appreciable responses and therefore the experimental LOD is approximately 6,000 cells. These results are shown in FIG. 18F.

[0269] Detection of CTSB in Cell Lysates Via Commercial CSTB Probe. 100 nM CTSB DLPBs were incubated with 15 pL of the cell line lysates (HT29, NCI-H508, RKO, SW-620) (~2 million cells / mL) to support the presence of active CTSB in the cell lysates. It was observed that all cell lines showed considerable response with the commercial CTSB substrate, supporting that there is active CTSB in the cell lysates. These results are shown in FIG. 18G. [0270] CTSB Inhibitor Studies'. To confirm that the response observed by incubating commercial CTSB and CTSB DLPBs comes from the activity of the CTSB protease, rather than the presence of, a commercial CTSB inhibitor w as used. When incubating the substrates with cell lysates and inhibitor, if the response is from solely protease activity, the signal generation should dramatically decrease, and the activity⁷ of the proteases decrease.

[0271] CTSB Commercial Probe with Cell Lysates and CTSB Inhibitor'. Commercial CTSB fluorogenic substrates were incubated with SW-620 cell lysates, with and without CTSB inhibitor to confirm the presence of protease activity⁷ in the cell lysates. The fluorogenic substrate was incubated for 30 minutes with 3 pL of the SW-620 cell lysates and read at a final concentration of 1 nM and 100 nM and read via a plate reader. It was observed that the commercial CTSB substrate does not yield a significant response at 1 nm, but at 100 nM there is a considerable response from the cell lysates. Upon incubating 100 nM of the commercial probe with cell lysates and the commercial CTSB inhibitor, the signal decreases and is similar to the background. This supports that the species responsible for the signal response is specifically from the activity of CTSB protease. These results are shown in FIGS. 18H-18I.

[0272] CTSB DLPB with Cell Lysates and CTSB Inhibitor'. CTSB DLPBs were incubated with SW-620 cell lysates, with and without CTSB inhibitor to further confirm the response seen if due to CTSB protease activity in the cell lysates. The CTSB and control 3CL DLPBs were incubated for 30 minutes with 3 pL of the SW-620 cell lysates and read at a final concentration of 1 nM and read via a plate reader. The 3CL DLPBs were also incubated in this manor to serve as a baseline non-specific activation/protease cleavage due to the cell lysates. It w as observed that the signal generated from the CTSB DLPB was increased relative to the 3CL DLPB control, indicating the detection of CTSB in the sample. It is also observed that the inclusion of the CTSB inhibitor decreases the response seen to the background and therefore supports the detection of CTSB activity in the cell lysates. These results are shown in FIG.

18J

Example 2: Peptide-DNA Hairpin-Based Sensor System

[0273] Disclosed herein is a peptide-DNA hairpin-based sensor system for the detection and monitoring of protease activity. The system utilizes a unique combination of a peptide- DNA hairpin structure and a CRISPR-Casl2a activation mechanism to provide a sensitive and accurate method of assessing the presence and activity of various proteases at room temperature.

[0274] The key component is a peptide-DNA hairpin structure that comprises a variable peptide sequence (3-50 amino acids in length) within the hairpin loop. This sequence acts as a recognition site for a target protease. When the protease of interest is in proximity, it cleaves the peptide sequence, causing the hairpin structure to break down into an intermolecular DNA double strand. This process results in a change in the DNA melting temperature and the structure further breaks down, releasing a short 8-base blocking DNA from a longer 24-base signaling DNA. Upon release, the signaling DNA strand is free to activate a CRISPR-Casl2a system, which subsequently cleaves a DNA reporter molecule. Since the hairpin structure does not activate the CRISPR-Casl2a system before cleavage, the presence of the free singlestranded signaling DNA (ssDNA) can be monitored as a proxy for protease activity⁷. Importantly, the release ssDNA can be detected via any DNA monitoring technique - not just CRISPR.

[0275] The primary purpose of the invention is to provide a highly sensitive and specific means of detecting and monitoring the activity of target proteases. This peptide-DNA hairpinbased sensor has various applications, including:

[0276] 1) Biomedical research: The sensor can be utilized in the study of proteases and their roles in physiological and pathological processes, such as inflammation, tissue remodeling, and cancer.

[0277] 2) Drug discovery: The invention can be employed in the identification and validation of protease inhibitors as potential therapeutic agents for various diseases and conditions.

[0278] 3) Diagnostics: The sensor system can be adapted for the development of rapid and sensitive diagnostic assays for the detection of specific proteases associated with certain diseases or pathological conditions.

[0279] The peptide-DNA hairpin sensor system is comprised of the following key physical, chemical, and biological components: [0280] 1) Peptide-DNA hairpin structure: A DNA-based hairpin structure containing a variable peptide sequence within its loop, acting as a recognition site for target proteases. The hairpin structure is designed to be stable under physiological conditions and stable under a range of salt concentrations and temperatures. This peptide should be between 3-50 amino acids.

[0281] 2) Signaling DNA: A 24-base single-stranded DNA molecule. Upon protease- mediated cleavage of the peptide sequence, the signaling DNA is released and can activate the CRISPR-Casl2a system.

[0282] 3) Blocking DNA: An 8-base single-stranded DNA molecule, which initially binds to and inhibits the signaling DNA. Upon protease-mediated cleavage, the blocking DNA is released.

[0283] 4) CRISPR-Casl2a system: A molecular machinery composed of the Casl2a endonuclease and a guide RNA molecule. Upon activation by the signaling DNA, the CRISPR- Casl2a system cleaves a DNA reporter molecule, allowing for detection.

[0284] 5) The invention harnesses the physical, chemical, and biological properties of these components to enable a sensitive and specific protease detection system. The peptide-DNA hairpin structure ensures target specificity, while the CRISPR-Casl2a activation mechanism allows for a robust and easily detectable readout of protease activity.

[0285] The invention of peptide-DNA hairpins is unusual in that it combines two different molecular structures, peptides, and DNA. into a single probe design. Compared to previous methods for detecting protease activity', which mainly rely on detecting the presence of peptide fragments or fluorescence generation through fragmentation, peptide-DNA hairpins offer several advantages. One key advantage is that they produce a free signaling DNA molecule when the peptide is cleaved by a protease. This free DNA molecule can then be used for downstream amplification, enabling the detection of protease activity with potentially higher sensitivity then what is currently possible. By designing hairpins with different peptide linkers that are cleaved by specific proteases, the detection of protease activity can be targeted towards a particular protease without the need for drastic changes in the entire probe structure.

[0286] Peptide-DNA hairpins solve a need for the sensitive and accurate detection of protease activity in solution. Traditional methods for detecting protease activity, such as mass spectrometry, can be expensive and time-consuming, making them impractical for routine use. Other methods, such as active site protease binding probes, and fluorophore quencher-based peptides, have limitations such as limited sensitivity or that they require the need to synthesize specific probes for each protease. Moreover, because of the inclusion of DNA, the strategy affords unparalleled multiplexing capabilities.

[0287] The peptide-DNA hairpin sensor offers several advantages over cunent technologies, making it a valuable innovation in protease detection:

[0288] 1) Specificity: The variable peptide sequence in the hairpin loop allows for targeted recognition of proteases of interest. This feature reduces the chances of cross-reactivity or false positives, which can be an issue with existing detection methods.

[0289] 2) Sensitivity: The ability to activate the CRISPR-Casl2a system after protease- induced cleavage significantly amplifies the signal, enabling the detection of even low concentrations of the target protease. This high sensitivity is particularly beneficial in scenarios where protease activity is low or subtle changes need to be monitored.

[0290] 3) Reduced background signal: The hairpin structure does not activate the Casl2a system before cleavage, which minimizes the background signal and improves the signal-to- noise ratio. This feature enhances the overall reliability of the assay.

[0291] 4) Versatility: The peptide-DNA hairpin sensor can be easily adapted for different proteases by altering the peptide sequence within the hairpin loop. This flexibility makes the technology broadly applicable across various research and diagnostic contexts.

[0292] 5) Simplicity⁷: The sensor design and its mechanism of action are relatively straightforward, reducing the complexity of the detection process. This simplicity facilitates the development of rapid and cost-effective protease detection methods.

[0293] 6) No expensive instrumentation required: Unlike mass spectrometry-based techniques, the Peptide-DNA hairpin sensor does not require costly and specialized equipment for detection. This advantage makes the technology more accessible and cost-effective, particularly for smaller labs and resource-limited settings.

[0294] 7) Consistent detection efficiency: In mass spectrometry techniques, variable peptide substrates can change their ionization efficiency, leading to alterations in detection limits for different proteases. The peptide-DNA hairpin sensor overcomes this challenge, as its mechanism is not dependent on ionization efficiency. This consistency ensures reliable protease detection regardless of the specific target protease.

[0295] 8) Reduced sample preparation: Mass spectrometry-based methods often necessitate extensive sample preparation and cleanup procedures, which can be timeconsuming and labor-intensive. The peptide-DNA hairpin sensor simplifies the detection process, allowing for faster and more streamlined protease detection workflows. [0296] 9) Potential for real-time monitoring: The peptide-DNA hairpin sensor's activation of the CRISPRCasl2a system and subsequent cleavage of a DNA reporter can potentially be adapted for real-time monitoring of protease activity. This capability can provide valuable insights into dynamic biological processes, which may be challenging or impossible to achieve with mass spectrometry based techniques.

[0297] 10) Operation at room temperature: The peptide-DNA hairpin sensors operate at room temperature unlike many other assays.

[0298] One way to overcome degradation by DNases is to modify the peptide-DNA hairpins to make them more resistant to enzy matic degradation. For example, researchers could add chemical modifications to the DNA strand that make it more stable and resistant to degradation. To overcome the effects of temperature, researchers could optimize the design of the peptide-DNA hairpins (adjust length of the peptides sequence, ssDNA sequence, and blocking strand length) to make them more stable under a range of conditions.

[0299] Additionally, the use of sequencing could enable the multiplexing of peptide-DNA hairpins, allowing for the simultaneous detection of multiple targets in a single assay. This could greatly enhance the efficiency and accuracy of diagnostic testing and may have applications in fields such as personalized medicine and drug discovery.

Example 3: Extending Peptide-DNA Hairpins into Ultra Sensitivity with True Multiplexation Capabilities

[0300] Proteases can trigger DNA hybridization of cleaved DLBPs. This proposed scheme is shown in FIG. 19A. As such, primer binding and amplification may be dependent on hairpin conformation. Similarly, proteases can trigger amplification and signaling DNA synthesis as shown in FIG. 19B. This method can use a polymerase buffer (e.g., 50 mM KC1, 2mM MgCh, IpM DNA Concentration), a blocked reverse primer (e.g., GGT ATC TAG ACG (SEQ ID NO: 16), which has T_m of 64°C), a primer (e.g., CGT CTA GAT ACC (SEQ ID NO: 17), which has T_m of 43°C), a reverse detection sequence (e.g., GTC TAA TAG GTA TCT AGA CG (SEQ ID NO: 18), which has T_m of 55°C), and a mock harpin (e.g., CGT CTA GAT ACC ATT TTT Because the initial DNA concentration is doubled each cycle, starting at I pM DNA will yield approximately 30,000 pM DNA after 15 cycles. The cleaved DNA can be amplified in many ways, for example, rolling cycle amplification or generating ssDNA from dsDNA.

[0301] Multiple hairpins can be amplified simultaneously in this way using unique barcodes for detection of each hairpin. This allows the scalable ability to detect proteases in one pot. The amplified sequences can be detected by, for example, detecting differences in DNA mass via gel electrophoresis, reading DNA sequences via TaqMan probes, or reading DNA sequences via NGS. Dyed nucleotides could also be used for detection. Such DNA microassays could be implemented for large scale multiplexing of many proteases.

[0302] FIG. 20 shows structural variations to the DLBPs.

Example 4: Conformationally Locked Peptide-DNA Nanostructures for CRISPR- Amplified Activity-Based Sensing

[0303] Fluorescent probes, such as molecular beacons (MBs), have revolutionized biomolecule detection, enabling significant advancements in diagnostic technologies [1-3], MBs typically function through a hairpin-shaped DNA structure that, when hybridized with a complementary target nucleic acid, separates a fluorophore-quencher pair, leading to a detectable increase in fluorescence signal. This mechanism has made molecular beacons an essential tool in nucleic acid detection, finding applications in cancer diagnostics, infectious disease monitoring, and personalized medicine [4-7],

[0304] Seitz, Grossmann, and others, have extended the utility of this technology⁷ to protein detection through the development of peptide beacons [8-13], These modified probes comprise a peptide in the loop region of the hairpin that changes conformation in response to protein binding, enabling the detection of protein biomarkers rather than nucleic acids. This innovation significantly broadens the scope of molecular beacons, allowing for their application in proteinbased diagnostics, thereby opening new avenues in early disease detection [8],

[0305] A key limitation of these beacon-type structures is the lack of a handle for signal amplification. In most cases, the interaction between the probe and the target is binding-based and occurs in a 1 :1 ratio which makes detecting low concentrations of targets challenging, thereby limiting the sensitivity of the assay. Additionally, binding-based probes are unable to distinguish between the active and inactive forms of biomolecules, which is a critical drawback in applications where only the active form is biologically relevant.

[0306] Activity-based sensing offers a powerful alternative to traditional binding-based methods, significantly enhancing diagnostic potential [14-16], This study presents the development of a new class of chemical probes — Cleavable, Locked Initiator Probes (CLIPs). These probes include a DNA-peptide-DNA triblock, conformationally locked into a hairpin nanostructure that resembles MBs but enables activity -based sensing and incorporates a handle for CRISPR-amplified signal generation. CRISPR-based sensing is chosen due to its ease of use and ability to detect targets at sub-picomolar levels under isothermal conditions [17-21], [0307] The study shows the functionality⁷ of CLIPs for detecting active proteases. The study focuses on proteases because of three primary reasons. First, they are often expressed in inactive precursor forms and are only activated under specific physiological conditions. Therefore, probes that can selectively detect the active form are required. Second, dysregulation of protease activity has been shown to be linked to various conditions, such as cancers [15,22,23], infectious diseases [24], neurodegenerative disorders [25,26], inflammatory conditions [27], and cardiovascular diseases [28], Activity' -based protease sensing has shown great promise in improving diagnostic and prognostic tools for these conditions [15,16,22-24,29-35], Third, detecting active proteases presents significant challenges. Traditional methods use peptide substrates that are labeled with reporters and can be cleaved by proteases of interest after which, the fragments are monitored through spectroscopy or mass spectrometry [36,37], While these methods can indicate protease activity, they come with several limitations. They often require elevated temperatures, suffer from limited sensitivity’, involve extensive sample processing, and depend on advanced instrumentation. Additionally, they typically necessitate high concentrations of both the substrate and the protease to generate a detectable signal, which diminishes their effectiveness in detecting loyv-abundance or weakly active proteases. This issue becomes especially critical in biological contexts, where local protease concentrations can be relatively high (up to ~mM) [38], but their levels in biofluids such as serum are much lower (~pM-nM) [39,40], making detection even more challenging. Consequently, more sensitive methods are needed to address these complexities in protease detection in clinical and diagnostic settings.

[0308] It has been previously shoyvn that CRISPR-based approaches can enhance the detection of active proteases [41-43], However, there are still key challenges that remain. Some methods require complex and costly synthetic biology, yvhich necessitates specialized expertise [43-45], Several approaches need elevated temperatures (above room temperature) for operation [43-45], Recently-developed nanoparticle-based approaches necessitate a separation step to differentiate between intact and cleaved peptides, adding complexity and increasing the risk of losing cleaved substrates [41,42,46], Furthermore, peptides presented on nanoparticle surfaces can be sterically hindered, limiting their accessibility to proteases. To overcome this, longer peptides (>10 amino acids) or spacers are often required to ensure efficient cleavage, complicating sensor design and reducing performance [41,47], The CLIP structure introduced herein overcomes these limitations, enabling one-pot detection of proteases at room temperature with commonly available instrumentation such as plate readers.

Results and Discussion

[0309] Design and Structural Characterization of CLIPs: The CLIP structure comprises three key components (FIGS. 22A-22C): (1) a peptide domain that is specifically recognized and cleaved by the target protease, (2) an “initiator’" DNA sequence that is capable of activating a CRISPR-Casl2a ribonucleoprotein (RNP) complex [17,20], and (3) a short “blocking” DNA strand. This blocking strand hybridizes with the initiator sequence to form a locked, hairpin-like nanostructure with a melting temperature (T_m) significantly above room temperature, ensuring that the probe remains inactive until specific protease-mediated cleavage occurs.

[0310] It was hypothesized that this locked state would sterically hinder the CRISPR- Casl2a ribonucleoprotein (RNP) complex from interacting with the initiator strand, thereby preventing signal transduction in the absence of active proteases (FIGS. 7A-7B and FIGS. 23A-23D). Upon cleavage of the peptide by the target protease, the probe undergoes a conformational shift from an intramolecular DNA duplex (locked state) to an intermolecular DNA duplex (unlocked state). This structural change lowers the T_m of the duplex, causing the blocking strand to spontaneously dissociate from the initiator strand (FIG. 1A). The freed initiator DNA (also referred to herein as “activator DNA”) then activates the CRISPR-Casl2a RNP complex, which, in combination with fluorophore-quencher labeled DNaseAlert™ reporters, generates an amplified fluorescence signal (FIG. IB), significantly enhancing the detection of active proteases [17,20],

[0311] As a proof-of-concept, the study created a CLIP for detecting the 3-chymotrypsin- like main protease (3CL) of SARS-CoV-2. 3CL was chosen as the model protease because it is relevant for detecting active SARS-CoV-2 infections and has a well-defined substrate [48— 50], The peptide sequence (TABLE 1), CSAVLQ(SGFK(N₃) (SEQ ID NO: 1, where | indicates the cleavage site), was modeled after a commercially available 3CL substrate [51] and was modified with a C-terminal azido-lysine. This modified peptide was chemically linked to a 24-nt initiator sequence (TABLE 2 and TABLE 3) equipped with dibenzocyclooctyne (DBCO) at the 5’ end, using click chemistry. The final DNA-peptide-DNA triblock structure was synthesized by adapting a recently developed proximity-based approach (FIG. 24) [52] . It is noted that alternative synthetic strategies, such as bead-based approaches could also be used to synthesize such triblock structures [53],

[0312] The desirable length of the blocking DNA was determined computationally using the IDT OligoAnalyzer™ tool (FIGS. 6A-6E, TABLE 4, TABLE 5, TABLE 6, and TABLE 11). Specifically, the study created hairpins composed entirely of DNA, with contour lengths similar to those of the CLIPs, and then calculated their T_m. Additionally, the study computed the Tm of just the stem portions to simulate the structure after proteolytic digestion of the CLIPs. The computational screening of 264 different conditions, including varying DNA concentrations, ionic strengths, and different stem and loop sizes, indicated that an 8-nt blocking DNA is ideal. This length would allow the CLIP to form a stable hairpin structure at 25°C and to spontaneously separate into single-stranded DNA after proteolytic cleavage, facilitating detection.

TABLE 11. Tm analysis of DNA hairpins of the form IzrXioBn and I24X30B11. The top section presents the T_m of DNA hairpins at 20 mM Mg²⁺ concentrations. The lower section shows the T_m of DNA hairpins at 40 mM Mg²⁺ concentrations. T_m values are expressed in degrees Celsius. Calculations were performed using IDT Oligo Analyzer™ at 100 mMNa⁺. DNA concentration was kept at 1 nM. NH indicates that a desirable hairpin structure is not formed.

[0313] The synthesized CLIPs were characterized using MALDI-MS (TABLE 9 and TABLE 10), UV-vis spectroscopy (FIGS. 25A-25B and FIG. 26), and gel electrophoresis (FIGS. 27A-27C). To confirm that proteases can cleave CLIPs, the study incubated 3CL-CLIP with excess 3CL protease overnight and analyzed the sample using MALDI-MS (FIG. 2A). The intact 3CL-CLIP displayed a dominant peak near its expected mass (calculated m/z: 12.569). After treatment with the protease, the probe showed two new peaks, which correspond to the lower-mass fragments produced by the cleavage of the CLIP (calculated m/z: 4,273 and 8,313). The cleavage was further supported by gel electrophoresis data (FIGS. 27A-27C).

[0314] The hairpin-like structure of the CLIP was confirmed by experimentally determining its T_m (FIG. 2B). The T_m of 1 pM of the CLIP was found to be ~53°C, similar to that of a DNA hairpin of comparable size (TABLE 4, TABLE 5, TABLE 6, and TABLE 11). This similarity suggests that the CLIP retains its intended locked structure in solution at room temperature. After treatment with protease, however, the T_m of the CLIP dropped to around ~25°C, which matches the Tm observed for a simple 1: 1 mixture of the initiator and blocking strands at the same concentration. This lower Tm indicates that the probe's stability decreases following protease-mediated cleavage.

[0315] CLIPs are Activated by Target Proteases: The study next investigated the functionality of CLIPs in reporting active proteases at room temperature (FIGS. 11B-11C, FIGS. 16A-16F, FIGS. 16I-16J, FIGS. 27A-27C, and FIG. 28). After incubating the CLIPs with 500 nM 3CL protease for 30 min. the study performed a CRISPR assay and detected a rapid increase in fluorescence within 5 minutes, yielding results within as quick as 35 min of total assay time. The fluorescence generated w as time-dependent and reached a plateau by 3 hours (FIG. 2C). In contrast, the fluorescence of the probes alone remained largely unchanged. Importantly, the DNA-peptide-DNA triblock structure is essential for seeing this locked behavior. For comparison, DNA-only hairpins with similar loop sizes and stem lengths activate CRISPR (FIGS. 11D-11E, FIGS. 12A-12C, FIGS. 13A-13B, and FIG. 29), suggesting that the initiator sequences in these hairpins remain accessible to the RNP, potentially through strand displacement (FIGS. 14A-14B, FIG. 15, and FIGS. 30A-20C). This finding underscores the structural uniqueness of CLIPs as conditional CRISPR activators, highlighting their potential utility in a variety of applications.

[0316] Next, the study benchmarked 3CL-CLIP against a commercial fluorogenic probe (3CL-CFP). 3CL-CFP is a rhodamine-labeled peptide in which the fluorescence of the rhodamine dye is quenched upon conjugation to the peptide. Cleavage by 3CL protease restores fluorescence, resulting in a turn-on signal. Compared to 3CL-CFP, 3CL-CLIP generated over 1.5-fold greater signal at 100 times lower concentration (FIG. 2D). This significant enhancement is attributed to the integration of DNA into the probe's design (FIG. 9B, FIG. 10, FIG. 31, FIG. 32. and FIG. 33). The study confirmed this by constructing a 3CL-CLIP with a beacon-like structure, where the probe is labeled with a fluorophore at one end and a quencher at the other. Protease-mediated peptide cleavage separates these two elements, triggering a fluorescence signal (FIG. 10 and FIG. 32). These findings demonstrate that CRISPR-based signal amplification allows for protease detection at probe concentrations 100 times lower than those required for direct detection using a traditional beacon-like structure (FIG. 33)

[0317] To assess the versatility of CLIPs, additional probes were synthesized for the proteases cathepsin B (CTSB), matrix-metalloprotease 7 (MMP7), and caspase-3 (CASP3) (FIG. 3A). These proteases were chosen due to their significance to cancer biology. Specifically, CTSB and MMP7 are known for their involvement in tumor growth and metastasis [54-58] and CASP3 is essential in the process of apoptosis [59], Detecting these proteases is vital for enhancing understanding of cancer mechanisms and for the development of diagnostic platforms. When incubated with their respective proteases, a pronounced increase in fluorescence signal was observed, indicating the successful detection of the target proteases. (FIG. 3B)

[0318] CLIPs are Sensitive and Selective Protease Sensors: The study next evaluated the potential of CLIPs for detecting diagnostically relevant proteases, particularly focusing on CTSB due to its overexpression in various cancers [54,57,58,60], A calibration curve was constructed by incubating CTSB-CLIP with increasing concentrations of CTSB protease for 30 min and then monitoring the CRISPR assay signal for an additional 2 h (FIG. 4B). The limit of detection (LOD) was determined using the 3o/m method. The LOD was found to be ~88 pM. In contrast, the LOD obtained using 100-times higher concentration of a CFP for CTSB was 3.4 nM, which is ~40-fold higher (FIGS. 34A-34B). The mechanism of action for CTSB- CFP is similar to that of 3CL-CFP, except that it uses 7-amino-4-methylcoumarin as the dye. Notably, the LOD of CTSB-CLIP is slightly higher compared to the lower end of the detection range of commonly used ELISA assays for CTSB (1.6-82 pM) [61-65], However, ELISA assays are unable to distinguish between active and inactive forms of the protein, require multiple steps, elevated temperatures (37°C) and a total assay time of up to 5 h. Moreover, the LOD of CTSB-CLIP is w ell below the reported CTSB concentrations in the serum of colorectal cancer patients, which can reach up to 297 pM [39] .

[0319] To assess the selectivity of CTSB-CLIP, the study tested its response to various proteases at 20 nM, including CTSB, 3CL, MMP7, thrombin (THR), CASP3, and trypsin (TRP). Bovine serum albumin (BSA) was used as an additional control to ensure non-specific protein binding does not disrupt the locked hairpin-like structure of CLIPs. The results (FIG. 4C and FIGS. 27A-27C) confirmed that CTSB-CLIP is highly selective for CTSB compared to the other tested proteases and BSA.

[0320] CLIPs Detect Proteases in Complex Biological Matrices: The study next explored the application of CLIPs in biologically relevant settings. The study first assessed the stability of CLIPs in the presence of nucleases (FIG. 35) and cell lysates (FIG. 36). DNase I. which degrades for single- and double-stranded DNA was applied to 3CL-CLIP at varying concentrations. These results (FIG. 35) show⁷ that after 30 min of treatment, while the CLIP structure is degraded at high DNase I concentrations, it remains stable at DNase activity of 25 U/L or lower. This is more than double the activity of DNase I in human serum (~10 U/L), demonstrating the robustness of the CLIP structure under relevant scenarios [66], Similarly, CLIPs were found to show minimal degradation in cell lysates for up to 6 h (FIG. 36).

[0321] The study next investigated the functionality of CLIPs in complex biological media by testing them with cell lysates and 10% human serum. Specifically, the study introduced 3CL protease into lysates from the NCI-H508 cell line, a type of colorectal cancer cell line that does not naturally produce 3CL protease. Any detection of signal in the absence of the spiked 3CL protease can be considered background noise. It was observed that mixing the cell lysates with 3CL-CLIP resulted in some signal generation, which is likely from non-specific cleavage of the probe or disruption of the locked structure due to the complex nature of the sample. However, when a spike of 1 pM 3CL protease was added to the lysates, the signal increased substantially, confirming that CLIPs are still effective in detecting target active proteases within a complex cell lysate solution (FIG. 37A). Similar results were observed in 10% human serum (FIG. 38). While the addition of 3CL-CLIP to 10% serum produced a higher background signal compared to buffer, the signal increased significantly upon adding 1 pM 3 CL protease, demonstrating that the probe remains effective in this medium. These findings align with control experiments using 3CL-CFP, a probe with a similar peptide sequence to 3CL-CLIP. which also exhibited high background signal in 10% human serum (FIG. 38).

[0322] The study further explored whether CLIPs could detect endogenous proteases in biological samples, choosing CTSB as the target protease due to its known dysregulation in colorectal cancer [60,67-69], The study investigated four different colon cancer cell lines - HT29, NCI-H508, RKO, and SW-620 (FIGS. 5A-5D, FIGS. 39A-39B, and FIG. 40). The lysates were incubated with CTSB-CLIP and assayed using CRISPR (FIGS. 5A-5D and FIGS. 37B-37D). To assess background signal, 3CL-CLIPs were used. CTSB-CLIPs treated with cell lysates showed up to a 21-fold increase in fluorescence relative to the fluorescence of the RNP alone within 30 min (FIGS. 5A-5D and FIG. 37B). While some background fluorescence was observed with the 3CL-CLIP, the signal from CTSB-CLIP was substantially higher. Using the most responsive cell line, SW-620, the probe successfully detected active CTSB from as few as 6,000 cells (FIGS. 39A-39B). For context, this number is on the lower end of cell quantities typically extracted during fine needle biopsies in cancer diagnostics [70], The study validated the presence of CTSB in the cell lysates using a CTSB-specific CFP (FIG. 37C and FIG. 40). Additionally, the study introduced a commercial CTSB inhibitor to the RKO lysates, which led to a reduction in fluorescence signals from both the CTSB-CFB and CTSB-CLIP (FIGS. 37C- 37D). These experiments confirm the specificity of this detection method for active CTSB in complex biological samples. Materials and Methods

[0323] Peptide sequences used in this study are shown in TABLE 1. Nucleic acid sequences used in this study are shown in TABLE 2 and TABLE 3. FIGS. 23A-23D shows a size comparison of CRISPR components and CLIPs.

[0324] Hypothesized Interaction Between CLIPs and Cleaved CLIPs with CRISPR- RNP: It was hypothesized that the hairpin-like conformation of CLIPs prevents the activation of CRISPR-Casl2a ribonucleoprotein (RNP) complexes until cleaved by an active protease. Uncleaved CLIPs should not activate the CRISPR-Casl2a RNP for two reasons: 1) the Blocking DNA prevents the Initiator RNA from hybridizing with the gRNA in the RNP, and 2) Steric hindrance caused by the non-DNA portion in the loop region of the CLIP obstructs the enzyme from accessing the Initiator DNA.

[0325] Once the CLIPs are cleaved by target proteases, the newly liberated single-stranded Initiator DNA is free from these impediments and activates the CRISPR-Casl2a. This activated CRISPR-Casl2a then exhibits non-specific endonuclease activity', allowing it to catalytically cleave DNase Alert reporters, which results in a significant increase in fluorescence. This fluorescence is then measured as a proxy for the protease's activity. This is depicted in FIGS. 7A-7B.

[0326] Buffers Commonly Used in the Study: Below⁷ is a list of buffers commonly used in this study, along with their respective concentrations. Composition is excluded for buffers that were purchased from commercial vendors.

TABLE 12. Buffers commonly used in the study.

[0327] Synthesis, Purification, and Characterization of DNA: DNA sequences were synthesized at the 1 pmol scale through solid-phase DNA synthesis using a MerMade 6 automated DNA synthesizer (LGC Biosearch Technologies. Alexandria MN 56308). DNA was synthesized on solid support, specifically controlled pore glass (CPG) beads. Depending on the sequence, either universal CPG (Glen Research, UnySupport CPG-1000, Cat. No. 20-5040) or on 3‘ black hole quencher CPG beads (3'-BHQ-2 CPG, Cat. No: 20-5932-01) were used.

[0328] All DNA phosphoramidites and materials required for solid-phase DNA synthesis were purchased from Glen Research (dA-CE phosphoramidite, Cat. No: 10-1000-10), (dC-CE phosphoramidite, Cat. No: 10-1010-10), (dT-CE phosphoramidite, Cat. No: 10-1030-10), (DMF-dG phosphoramidite, Cat. No: 10-29-10), (DBCO-dT-CE phosphoramidite, Cat. No: 10-1539-95). (5 -DBCO-TEG Phosphoramidite, Cat. No: 10-1941-90). (Cyanine 5 Phosphoramidite, 10-5915-95). (0.25M 5-Ethylthio-lH-Tetrazole (ETT) in Anhydrous Acetonitrile, Cat. No: 30-3140-52), (3% TCA/DCM, Cat. No: 40-4140-57), (0.02M Iodine in Tetrahydrofuran/Pyridine/Water (70:20: 10), Cat. No: 40-4330-52), (Anhydrous acetonitrile, 40-4050-57). Sequences made this way include Initiator DNA, Blocking DNA, Quencher- labeled Initiator DNA, Dye-labeled Blocking DNA. Sequences purchased custom from Integrated DNA Technologies include DNA Hairpin 30-8, DNA Hairpin 10-8, 24-nt DNA blocking DNA, and 16-nt DNA blocking DNA.

[0329] After synthesis the beads were treated with 1 mL of a 30% ammonium hydroxide solution (Sigma-Aldrich, Cat. No. 221228-100ML-A) for 17 h at room temperature (RT). The solution was then evaporated under pressurized air at RT (for ~10 min). 1 mL water was then added, and the beads were separated from free DNA in solution using a syringe filter and then purified.

[0330] Unmodified DNA sequences (in which the desired full-length sequences contain a 4,4'-dimethoxytrityl group) or those containing 5 DBCO-TEG groups, were purified using a Glen-Pak cartridge (Glen Research, Glen-Pak DNA purification cartridge, Cat. No. 60-5200) following the manufacturer’s recommended protocol.

[0331] DNA containing a 3’DBCO-dT group, specifically the Blocking DNA, was purified through high performance liquid chromatography (HPLC) using a Vanquish HPLC (ThermoFisher Scientific, Vanquish Core HPLC Systems, Cat No: VQ-CORE-BIN-01). Buffer A was 30 mM triethylammonium acetate (prepared by mixing 1: 1 molar equivalents of triethylamine (Fisher Scientific, Triethylamine (Reagent). Cat No: 04885-1) and glacial acetic acid (Fisher Scientific, Acetic Acid, Glacial (TraceMetal Grade), Cat No: A507-P500) over ice to pH 7 with 3% acetonitrile (ACN) in water, while buffer B was 100% acetonitrile. The method followed a gradient increase from 10% to 35% buffer B over 80 min, a ramp to 100% buffer B over 5 min followed by 5 min of 100% buffer B, a ramp down to 5% buffer B over five min followed by 5% buffer B for 5 min. Separation was monitored through UV-Vis peaks at 260 nM and 310 nM for DNA and DBCO respectively. All peaks were collected, lyophilized, and redissolved in water for characterization.

[0332] Synthesis, Purification, and Characterization of Peptides: All peptides were synthesized in house using standard Fmoc-based solid-phase synthesis on a 0. 1 mmol scale. Synthesis was conducted using Rink Amide AM resin 100-200 mesh (Sigma- Aldrich, Cat. No. 183599-10-2) inside a fritted 6 mL syringe. The resin was initially prepared by washing with 5 mL of dimethyl formamide (DMF) (Millipore Sigma. N,N-Dimethylformamide. Cat No: 270547). for 15 min.

[0333] Peptides were synthesized by adding amino acids sequentially in the following cycle: (1) the functionalized resin was washed with 5 mL of dimethyl formamide (DMF). (2) Next, the sample was deprotected using 5 mL of 20% piperidine (Millipore Sigma. Piperidine Solution 20% in DMF. Cat No: 80645) for 5 min and the piperidine was removed through the syringe. Then, the same reagent was added and the deprotection step was continued for another 20 min. Piperidine was washed from the syringe using DMF until the piperidine smell disappeared. (3) The amino acid (0.25 mmol) was then added to the resin along with coupling reagents (2.5 equiv. HOBL 2.5 equiv. DIC) in 3 mL DMF. This coupling was run for 2 h at RT but couplings involving Fmoc Lys (Ns)-OH (AnaSpec.Inc, Cat. No. AS-53100-F1) were allowed to run overnight. This cycle was followed until all amino acids had been added and then deprotected using the earlier mentioned procedure.

[0334] Peptides were cleaved from the resin surface using 1 mL of a cocktail solution of trifluoroacetic acid (TFA) (Sigma-Aldrich, Cat. No. 302031-100ML), H2O, and triisopropylsilane (TCI, Cat. No. T1533) in a volumetric ratio of 95:2.5:2.5, shaking at RT for two h. Taking 100 pL portions of this solution and adding them dropwise into chilled diethyl ether (Millipore Sigma. Diethyl ethyl. Cat No:296082) the peptide was precipitated and recovered using centrifugation at 15,000 rpm for 3 min. This was repeated until all the solution was collected and was further washed using this method using fresh chilled diethyl ether. The peptide was then stored dry' at 4°C until further use.

[0335] Synthesis, Purification, and Characterization of CLIPs: CLIPs were synthesized sequentially as per the reaction scheme in FIG. 24. First, peptide-DNA conjugates were synthesized through copper free click chemistry using azido-peptides and dibenzocyclooctyne (DBCO) modified DNA. The peptides used were (Pep-3CL, Pep-MMP7, Pep-CTSB, and Pep-CASP3 from TABLE 1), and the DBCO-DNA was the Initiator DNA as seen in TABLE 2 and TABLE 3. Peptides were reacted with DNA at a 5: 1 molar ratio in IX phosphate buffered saline (PBS) (Thermo scientific, pH 7.4, Cat. No: J62036.K2) with shaking at RT overnight. 1 pmol of Initiator DNA was used and reacted at a concentration of ~ 600 pM. The reaction was monitored for completion via UV-Vis. Disappearance of the DBCO’s characteristic 310 nm peak indicated that all DNA had been reacted. The solution was then washed using a 3 kDa centrifuge filter (Amicon Ultra. UFC500396) five times with 0.5 mL of ultrapure water and characterized via UV-Vis.

[0336] Free peptides were removed from the peptide-DNA conjugates using a Macro-Prep DEAE weak anionic exchange resin (Bio-Rad. Cat. No: 158-0020). Into a fritted 6 mL syringe, 2 mL of the DEAE resin was loaded and washed with 5 column volumes of ultrapure water. Before applying the sample, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Sigma Aldrich, Tris(2-carboxyethyl) phosphine hydrochloride, Cat No: C4706) was added to the sample at a final concentration of 10 mM and allowed to shake at 1500 RPM (Benchmark, Multi-Therm Heating Shaker. Cat No: H5000-H) for 15 min to break any disulfide bonds between the peptides. The entirety of the sample was then applied to the DEAE at a flow rate of 1 drop per second, washed with 5 column volumes of water, then of 0.3 M NaCl, and peptide- DNA conjugates were then eluted with 1 mL of 1 M sodium chloride (NaCl) (Fisher Bioreagents, Sodium Chloride, BP358-212). The elution of the peptide-DNA conjugate was monitored using UV-Vis utilizing DNA s characteristic absorbance at 260 nm. No significant DNA peaks were seen in water or 0.3 M NaCl washes. However, when 1 mL of 1 M NaCl was used, the peptide-DNA conjugate could be recovered (-50%). Further washing with additional 1 M NaCl or higher concentrations did not yield additional DNA. The sample was then washed using a 3 kDa centrifuge filter five times with 0.5 mL of ultrapure water and peptide-DNA conjugate was characterized via UV-Vis, MALDI-MS, and gel electrophoresis. [0337] Next, an azido PEG linker was installed on the peptide-DNA conjugate. These linker-peptide-DNAs were synthesized though NHS-ester conjugation between the peptide- DNA conjugate and NHS-PEG4- Azide (ThermoFisher, Cat. No: 26130). Before reacting, the peptide-DNAs were treated with TCEP at a final concentration of 10 mM shaking for 15 min. NHS-ester conjugation was carried out using 0.5 pmol of the peptide-DNAs and a 150-molar excess of the NHS-PEG4- Azides in 2 mL of sodium bicarbonate (Fisher Bioreagents, Sodium Bicarbonate. Cat No: BP328-500) with shaking at RT overnight. The sample was then washed using a 3 kDa centrifuge filter five times with 0.5 mL of ultrapure water and final conjugate was characterized via UV-Vis. Final yield was on average was -85%.

[0338] The final CLIP structure (i.e. the DNA-peptide-DNA triblock) was synthesized by reacting linker-peptide-DNAs with Blocking DNA using copper free click chemistry. Then, 0.1 pmol of the linker-peptide-DNA was conjugated to the Blocking DNA (5: 1 molar excess of Blocking DNA relative to linker-peptide-DNA) in 0.1 M NaCl PBS with shaking at 1500 RPM at RT for 16-48 h. The sample was then washed using a 3 kDa centrifuge filter ten times with 0.5 mL of ultrapure water and final conjugate was characterized via UV-Vis, MALDI- MS, and gel electrophoresis.

[0339] Purification of CLIPs was accomplished using HPLC separation. Buffer A was 30 mM tri ethylammonium acetate buffer pH 7 and 3% acetonitrile (ACN) in water, while buffer B w as 100% acetonitrile. The method followed a gradient increase from 10% to 25% buffer B over 80 min, a ramp to 100% buffer B over 5 min followed by 5 min of 100% buffer B, then a ramp down to 5% buffer B over five min followed by 5% buffer B for 5 min. All peaks were collected, lyophilized, and redissolved in water for characterization via MALDI-MS. For the purification of the caspase-3 (CASP3) CLIP, a similar method w as used but the initial gradient was increased from 13% to 27% buffer B over 80 min.

[0340] Synthesis, Purification, and Characterization of FQ-3CL-CLIP: Synthesis of a fluorophore-quencher (FQ) 3CL-CLIP was accomplished using a similar procedure to regular CLIPs with a few differences described herein. In place of the Initiator DNA, a Quencher- labeled Initiator DNA containing a 3‘ black hole quencher (BHQ) was used. In place of Blocking DNA, Dye-labeled Blocking DNA containing a 5’ cyanine 5 dye was used. Care was taken to ensure the materials w ere stored in dark conditions as well as covered during handling to decrease the chance of them being damaged by ambient light. All conjugation chemistry steps were the same as those used for CLIPs except that the conjugation of linker-peptide- Quencher-labeled Initiator DNA to Dye-labeled Blocking DNA was done over 72 h in a 1: 1 ratio. The final product, FQ-3CL-CLIP, was washed using a 3 kDa centrifuge filter 10 times with 0.5 mL of ultrapure water, purified via gel electrophoresis, and characterized via MALDI- MS.

[0341] UV-Vis Characterization: UV-Vis spectroscopy (Agilent, Cary 60 UV-Vis) was used to characterize DNA concentrations, the presence of DNA modifications (e.g., DBCO, dye, quencher, etc.), as well as completion of copper-free click chemistry . A standard 1 cm pathlength cuvette was used.

[0342] MALDI-MS Characterization: MALDI-MS characterization of DNA, peptides. peptide-DNA conjugates, linker-peptide-DNAs, and CLIPs was accomplished using a Broker AutoFlex Max MALDI-TOF instrument. The matrix utilized for all samples contained 2', 6'- dihydroxyacetophenone (DHAP) (Sigma-Aldrich, Cat. No. 37468). The matrix was prepared by dissolving 25 mg of the DHAP in 333 pL of methanol (Fisher Chemical, Methanol (HPLC Grade), Cat No: A452Sk4) to which 111 pL of saturated ammonium citrate (Sigma Aldrich, Ammonium Citrate Dibasic, Cat No: 247561) was added dropwise. A white precipitate was immediately seen and allowed to settle for 15 min. The clear yellow liquid supernatant was used as the matrix. When plating for MALDI-MA, 2 pL of sample was added followed by 1 pL of the DHAP matrix. This resulted in rapid crystallization and was allowed to air dry. ~ 2 min, before MALDI-MS.

[0343] To examine the protease cleavage of CLIPs, 1 pM of the 3CL-CLIP was treated with 2 pM 3CL protease (Sigma Aldrich, Cat. No: SAE0172-200UG) overnight in 3CL Protease Assay Buffer (BPS Bioscience, Cat. No: 79956). with shaking at 1500 rpm at RT. This process took place in a total volume of 100 pL. Subsequently, the solution was washed ten times using a 3 kDa centrifugation filter with 0.5 mL of ultrapure water to remove any salts. The sample remaining in the filter (approximately 10 pL) was then characterized via MALDI- MS.

[0344] PAGE Gel Analysis of CLIPs :

[0345] Characterization and Purification of CLIPs. Native polyacrylamide gel electrophoresis (PAGE) was utilized to reinforce the MALDI-MS characterization of DNA, peptide-DNA conjugates, linker-peptide-DNAs. and CLIPs. The 15% acrylamide gels, which were produced in-house, were made by diluting a stock 40% acrylamide solution (Bio-Rad. 40% Acrylamide/Bis Solution 19: 1, Cat no: 1610144) with IX Tris-Borate-EDTA buffer (TBE). The IX TBE solution was created by diluting 10X TBE (Fisher BioReagents, Tris- Borate-EDTA 10X, Cat No: BP1333-4) with deionized water.

[0346] 50 pL N.N,N’.N^?-Tetramethyl-ethylenediamine (Sigma Aldrich, N.N,N’.N^?-

Tetramethyl-ethylenediamine, Cat No: T9281) and ~2 mg of ammonium persulfate (Fisher BioReagents, Ammonium Persulfate (APS) Electrophoresis, Cat no: BP 17) were added to the 15% acrylamide solution to initiate gelation. After mixing well, this solution was poured into a Mini-PROTEAN Tetra Handcast System (Bio-Rad, Cat. No: 1658050). A ten-well comb was placed at the top of the gel, which was allowed to solidify. Following the removal of the comb, the gel wells were thoroughly rinsed with water. The gel was then installed in a Mini- PROTEAN Tetra System (Bio-Rad, Cat No: 1658005EDU) and filled to the manufacturer's recommended line with IX TBE buffer.

[0347] For PAGE characterization, all samples (DNA, peptide-DNA, linker DNA, and CLIPs) were prepared at a final concentration of - 1 pM with a total volume of 15 pL. These samples contained 2 pL of glycerol. In addition, a sample containing ultralow DNA molecular weight ladder (Invitrogen, Tracklt Ultra Low Range DNA Ladder. Cat No: 10488023) was also prepared. The ladder sample was prepared by adding 2 pL of glycerol to 0.2 pL of the ladder stock solution and diluting to a final volume of 15 pL with TBE. 10 pL of all samples were loaded into different wells of the prepared 15% PAGE gel. To track gel progression, 10 pL of a gel loading dye (Sigma Aldrich, Gel Loading Solution, Cat No: G7654) was added into a separate dedicated well.

[0348] The gel was run at a voltage of 120 V until the loading dye had traveled -90% of the gel's length, at which point it was stopped. The gel was then removed from the holder, placed in approximately 50 mL of water and stained with GelRed nucleic acid stain (Milipore Sigma. GelRed Nucleic Acid Stain (10.000X, Water), Cat No: SCT123) for 10 min. Finally, the gel was placed into a Bio-Rad ChemiDoc MP imaging system (Bio-Rad, Cat No: 12003154), and the GelRed stain was monitored at Ex: 590 Em: 110.

[0349] Denaturing PAGE was used to purify FQ-3CL-CLIP. As before, a 15% PAGE gel was created. However, the gel also contained 8 M urea (Fisher Chemical, Urea, Cat No: U15- 500). The conditions for running the gel w ere the same as mentioned above.

[0350] For purification, 20 nmol of the FQ-3CL-CLIP was purified via gel electrophoresis. Individual bands could be visualized due to the presence of cyanine 5 dye and BHQ. These bands were extracted from the gel using a razorblade and placed into 15 mL Falcon tubes. The bands were then crushed using a pipette tip, and 3 mL of deionized water was added to each tube. The mixture was then frozen using liquid nitrogen and thawed three times to aid in the crushing of the gel. Thereafter, the sample was placed on a tube rotator for 48 h. At this point it w as noted that the intensity of the color of the gel decreased and that of the surrounding solution increased, indicating that the colored chemical species had migrated into the water. The gel fragments were separated via centrifugation and passage through a fritted syringe, and the remaining solution was washed 10 times using a 3 kDa spin filter to remove excess urea. The solution was then characterized via UV-Vis and the presence of purified FQ-3CL-CLIPs were confirmed via MALDI. The approximate yield of this separation was 30%, exceeding what was achieved via HPLC. However, MALDI-MS indicated that this sample was slightly more impure compared to the HPLC-purified CLIPs.

[0351] Time-dependent Protease-Mediated Cleavage of CLIPs. Time-dependent cleavage of CLIPs was studied using denaturing PAGE gel. 1 pM FQ-3CL-CLIP was prepared in 3CL buffer with 1 pM 3CL protease. Samples were incubated for 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h. At each point, 2 pL of the solution was mixed with 8 pL of 8 M urea to stop the reaction. A control containing 1 pM FQ-3CL-CLIP without protease was also prepared. Next, 2 pL of glycerol was added to all samples, including the control. Samples were loaded onto a 15% denaturing PAGE gel. To track gel progression, 10 pL of gel loading dye (Sigma Aldrich, Gel Loading Solution, Cat No: G7654) was added into a separate well. The gel was run using the same procedure as for native PAGE and monitored in the Cy5 channel.

[0352] To study the selectivity of CLIPs, 1 pM FQ-3CL-CLIP was prepared in 3CL and CTSB buffers, spiked with 1 pM 3CL protease and 1 pM CTSB protease, respectively. After incubation for 2 h and 4 h, 2 pL of each solution was mixed with 8 pL of 8 M urea to stop the reaction. A control containing 1 pM FQ-3CL-CLIP without protease was also prepared. Next, 2 pL of glycerol was added to all samples, including the control. Samples were loaded onto a 15% denaturing PAGE gel. To track gel progression, 10 pL of gel loading dye (Sigma Aldrich, Gel Loading Solution, Cat No: G7654) was added into a separate well. The gel was run using the same procedure as for native PAGE and monitored in the Cy5 channel.

[0353] Stability) of CLIPs in Cell Lysates'. To study the stability of CLIPs in cell lysates, 1 pM FQ-3CL-CLIP was prepared in PBS and incubated with 5 pL of lysates from the colorectal cancer cell line RKO (CRL-2577) in a total volume of 15 pL. At 30 min, 1 h, 3 h, and 6 h incubation times, 2 pL of the solution was mixed with 8 pL of 8 M urea to stop the reaction. A control containing 1 pM FQ-3CL-CLIP without protease was also prepared. Next, 2 pL of glycerol was added to all samples, including the control. Samples were loaded onto a denaturing PAGE gel prepared as described previously. To track gel progression. 10 pL of gel loading dye (Sigma Aldrich, Gel Loading Solution, Cat No: G7654) was added into a separate well. The gel was run using the same procedure as for native PAGE and monitored in the GelRed and Cy5 channels.

[0354] Melting Temperature Analysis'. To verily the hairpin-like structure of intact CLIPs and the absence of such structures in protease-cleaved CLIPs, the study conducted melting assays on the following samples: 3CL-FQ-CLIP with and without 3CL protease. Dye-labeled Blocking DNA, and a 1 : 1 mixture of Dye-labeled Blocking DNA and Quencher-labeled Initiator DNA. When CLIPs are folded into a hairpin, close proximity between the fluorophore and the quencher suppresses the fluorescence signal. Dehybridization at higher temperatures opens the hairpin, increases the distance between these molecules, and results in greater fluorescence signal.

[0355] All samples were prepared at a concentration of 1 pM in 3CL buffer. To test the effect of protease cleavage on melting temperature, 1 pM of the FQ-3CL-CLI was incubated with 3 pM of 3CL protease in 3CL buffer overnight for running the melting assay.

[0356] Melting assays were conducted by dispensing 10 pL of samples into each well of a 96-well plate (Bio-Rad, Hard-Shell High Profile 96-Well Semi-Skirted PCR Plates. Cat. No: HSS9601), arranging these in triplicate. A qPCR instrument (Bio-Rad, CFX96 Real-Time System Cl 000 Touch Thermal Cycler) was used for the procedure. The qPCR method was as follows: The lid temperature was consistently maintained at 105°C throughout the assay. Initially, the samples were heated to 90°C over a period of 5 minutes and then held at this temperature for 30 seconds. After this, the samples were cooled to 4°C, where the initial fluorescence reading was recorded in the cyanine 5 channel. The temperature was then increased by 0.5°C increments, with readings taken at each increment in the selected channel. This cycle of increasing temperature and taking readings continued until the temperature reached 95 °C.

[0357] To account for any photobleaching effects, the fluorescence signal from the Dye- labeled Blocking DNA was used for correction. The study also ran additional controls including 3CL buffer, BHQ stand, and 1 pM 3CL protease; as expected, these showed no fluorescence in the cyanine 5 channel.

[0358] Fluorescence Assays:

[0359] Optimizing CRISPR-Cas 12a Assays: CRISPR-Casl2a ribonucleoprotein solution

(RNP) was formed using CRISPR-Casl2a enzyme Alt-R™ L.b. Casl2a (Cpfl) Ultra (Integrated DNA Technologies, Ref. No: 443255472), guide RNA purchased from Integrated DNA Technologies (TABLE 2 and TABLE 3). and reporter DNase alert (Integrated DNA Technologies, Cat. No: 11-04-03-03). These were prepared in a CRISPR Buffer. To ensure repeatability of CRISPR assays, stock guide RNA and CRISPR-Casl2a were aliquoted into single use tubes.

[0360] All fluorescence experiments were conducted using plate readers. Two types of plate readers were used: Agilent, BioTek Cytation 5 Imaging Multimode Reader and Agilent, BioTek Synergy Hl Multimode Reader. It should be noted that the gain between these plate readers is different and therefore RFU signals may differ between experiments.

[0361] The study first investigated how the concentration of Initiator DNA affects the fluorescence signal observ ed in the CRISPR assay. For this, different concentrations of the Initiator DNA (3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0 nM) was added to CRISPR buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). The fluorescence was measured using a plate reader. Samples were read at 25°C after 30 min using an excitation wavelength of 525 nm and an emission wavelength of 566 nm.

[0362] The study next investigated how the concentration of the RNP affects the fluorescence signal observed in the CRISPR assay. For this, the Initiator DNA (fixed at a final concentration of 1 nM) was added to CRISPR buffer containing the RNP at various concentrations. The RNP was a 1 :1 mixture of Casl2a and the gRNA. The concentrations of RNP used were 100 nM. 10 nM, 1 nM, and 0.1 nM. DNase Alert reporter was added to each well at a concentration of 2.5 uL/100 uL. The fluorescence was measured via plate readers. Samples were read at 25°C and shaken for 30 seconds before reading fluorescence every three min at an excitation wavelength of 525 nm and an emission wavelength of 566 nm.

[0363] Effect on Protease Incubation on the Ability of Initiator DNA to Activate CRISPR- Casl2a Signaling'. 10 nM Initiator DNA was incubated with and without 3 pM 3CL protease for 2 h with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final Initiator DNA concentration while reading fluorescence in the CRISPR assay was 1 nM.

[0364] CRISPR-Casl2a Assays with CLIPs: 10 nM CLIPs were incubated with varying concentrations of proteases for 30 min-24 h with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA. and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM.

[0365] Here is one example of how solutions were prepared for one assay: Solution 1 : 10 nM CLIPs were incubated with proteases in a total of 40 pL of protease-specific assay buffer for 30 min-24 h with shaking at 1500 RPM. Solution 2: A solution of 20 nM CRISPR-Casl2a, 20 nM gRNA, and 5.0 pL/well of DNase alert was prepared in CRISPR-Casl2a assay buffer. When testing protease activity , solution 1 and 2 were added to each well in a black 96 well plate in the following order, 40 pL of CRISPR buffer, 50 pL of Solution 2, and 10 pL of Solution 1.

[0366] Effect of CLIP Concentration on CRISPR-Cas 12a Assays: 3CL-CLIPs were serially diluted by 10-fold from 10 nM to 10 pM, incubated with 3 pM of 3 CL protease for 2 h, and then subjected to the CRISPR-Cas 12a assay.

[0367] Detection of 3CL, CTSB. MMP7, and CASP3 Proteases in Buffer: 10 nM CLIPs targeting various proteases were incubated with their respective proteases for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). The proteases used in this study were 3CL (Millipore Sigma, Mpro, 3CL Protease from coronavirus SARS-COV-2, SAE0172), CTSB (Millipore Sigma. Cathepsin B. Human Liver, 219362-50UG), MMP7 (Enzo, MMP-7 catalytic domain, human, recombinant, BML-SE181- 0010), and CASP3 (Enzo, Caspase-3 human, recombinant active, ALX-201-059-U025). Protease concentrations used were 500 nM for 3CL, 500 nM for CASP3. 250 nM for MMP7, and 20 nM for CTSB. Each protease was incubated with its corresponding CLIP in its appropriate assay buffer before being subjected to the CRISPR assay. After incubation, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence was measured via plate readers. Samples were read at 25°C and shaken for 30 seconds before reading fluorescence every 3-5 min at an excitation wavelength of 525 nm and an emission wavelength of 566 nm.

[0368] Determination ofLOD of 3CL-CLIP: To determine the limit of detection (LOD) for the 3CL-CLIP. 10 nM 3CL-CLIP was incubated with varying concentrations of 3CL protease in 3CL Buffer for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). The 3CL protease concentrations used were 500 nM, 250 nM, 125 nM, and 0 nM. Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final 3CL-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read over time using a plate reader (excitation wavelength of 525 nm and an emission at 566 nm). The fluorescence at the 30 min time point from the CRISPR assay was plotted at a function of 3CL concentration to form a calibration curve. From the initial slope of this calibration curve, the LOD was calculated using the 3o/m method. [0369] Comparison of 3CL-CLIP with DNA only Hairpins'. To investigate how CLIP hairpins compare with DNA-only Hairpins in their ability to activate CRISPR enzymes, the study performed several experiments.

[0370] The study first investigated how the addition of 24-nt and 16-nt Blocking DNA affects the CRISPR activating capabilities of Initiator DNA. For this, 1 nM of the Initiator DNA was treated with 10 nM of the 16-nt Blocking DNA or 24-nt Blocking DNA. Then CRISPR assays were run (final concentration of 10 nM by Casl2a. 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). The fluorescence was measured using a plate reader. Samples were read at 25°C after 30 min using an excitation wavelength of 525 nm and an emission wavelength of 566 nm.

[0371] The study next conducted CRISPR assays using Initiator DNA, DNA Hairpin 10- 8, DNA Hairpin 30-8, and CLIP, each at 1 nM final concentration in the CRISPR assay.

[0372] Following this, the study investigated how the addition of 24-nt and 16-nt Blocking DNA affects the CRISPR activating capabilities of DNA Hairpin 30-8. For this, 1 nM of the DNA Hairpin 30-8 was treated with 10 nM of the 16-nt Blocking DNA or 24-nt Blocking DNA. Then CRISPR assays were run (final concentration of 10 nM by Cast 2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). The fluorescence was measured using a plate reader. Samples were read at 25°C after 30 min using an excitation wavelength of 525 nm and an emission wavelength of 566 nm.

[0373] Comparison of 3CL-CLIP with 3CL-CFP'. To benchmark the protease sensing capabilities of 3CL-CLIP with commercially available probes, a fluorogenic substrate of 3CL (Bio-Techne, SARS CoV-2 3CL protease substrate Rhl 10-conjugated, Cat. No: S-720-200) was purchased. This probe is referred to as 3CL-CFP.

[0374] 10 nM 3CL-CLIP was incubated with 250 and 500 nM 3CL protease in 3CL Buffer for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final 3CL-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read using a plate reader after an additional 60 min (excitation wavelength of 525 nm and an emission at 566 nm). Therefore, total assay time is 90 min.

[0375] For comparison, 10 nM 3CL-CFP was incubated with 250 and 500 nM 3CL protease in 3CL Buffer for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10- fold with CRISPR Buffer. Therefore, the final 3CL-CFP concentration at fluorescence reading was 1 nM. The fluorescence from 3CL-CFP was read using a plate reader after an additional 60 min (excitation wavelength of 485 nm and an emission at 535 nm). Therefore, total assay time is 90 min. This assay was also performed using 1 pM of 3CL-CFP (final probe concentration at fluorescence reading is 100 nM).

[0376] Comparison of 3CL-CLIP with FQ-3CL-CLIP . To evaluate the extent of the amplification of the CRISPR-Cas l2a system provided on the CLIP signal, a comparison using a FQ-3CL-CLIP was performed.

[0377] 10 nM 3CL-CLIP was incubated with 250 and 500 nM 3CL protease in 3CL Buffer for 30 min with shaking (Benchmark, Multi -Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final 3CL-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read using a plate reader (excitation wavelength of 525 nm and an emission at 566 nm).

[0378] 1 pM FQ-3CL-CLIP was incubated with 250 and 500 nM 3CL protease in 3CL

Buffer for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer. Therefore, the final FQ-3CL-CLIP concentration at fluorescence reading was 100 nM. The fluorescence from FQ-3CL-CLIP read using a plate reader (excitation wavelength of 648 nm and an emission at 700 nm). This assay was not performed using 10 nM of FQ-3CL-CLIP (final probe concentration at fluorescence reading is 1 nM) as no fluorescence change could be seen at this concentration.

[0379] The study performed an additional test with FQ-3CL-CLIP to ensure that the 3CL protease can cleave it. For this, 100 nM FQ-3CL-CLIP was added to 1 pM 3CL protease in 3CL Buffer. The fluorescence from FQ-3CL-CLIP read using a plate reader (excitation wavelength of 648 nm and an emission at 700 nm) over time.

[0380] Comparison of CTSB-CLIP with Commercial CTSB Fluorogenic Substrate. To benchmark the protease sensing capabilities of CTSB-CLIP with commercially available probes, a fluorogenic substrate of CTSB (Sigma- Aldrich, Z-Arg-Arg-7-amido-4- methylcoumarin hydrochloride, Cat. No. C5429) was purchased. This probe is referred to as CTSB-CFP.

[0381] 10 nM CTSB-CLIP was incubated with 10 nM CTSB protease in CTSB Buffer for

30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final CTSB-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read using a plate reader after an additional 120 min (excitation wavelength of 525 nm and an emission at 566 nm). Therefore, total assay time is 150 min.

[0382] For comparison, 10 nM CTSB-CFP was incubated with 10 nM CTSB protease in CTSB Buffer for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer. Therefore, the final CTSB-CFP concentration at fluorescence reading was 1 nM. The fluorescence from CTSB-CFP read using a plate reader after an additional 120 min (excitation wavelength of 380 nm and an emission at 460 nm). Therefore, total assay time is 150 min.

[0383] Determination ofLOD of CTSB-CLIP and CTSB-CFP'. To determine the limit of detection (LOD) for the CTSB-CLIP, 10 nM CTSB-CLIP was incubated with varying concentrations of CTSB protease in CTSB Buffer for 30 min with shaking (Benchmark, MultiTherm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). The CTSB protease concentrations used were 30 nM, 20 nM, 15 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, and 0 pM. Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final CTSB-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read over time using a plate reader (excitation wavelength of 525 nm and an emission at 566 nm). The fluorescence enhancement at the 120 min time point from the CRISPR assay was plotted at a function of CTSB concentration to form a calibration curve. From the initial slope of this calibration curve, the LOD was calculated using the 3o/m method.

[0384] To determine the limit of detection (LOD) for CTSB-CFP, 1 pM CTSB-CFP was incubated with varying concentrations of CTSB protease (25 nM, 10 nM, 5 nM, 0. 1 nM, and 0 nM) in CTSB Buffer for 30 minutes at 25°C with shaking at 1500 RPM (Benchmark MultiTherm Heating Shaker, Cat No: H5000-H). After incubation, the samples were diluted 10-fold with CRISPR Buffer, resulting in a final CTSB-CFP concentration of 100 nM during fluorescence measurement. Fluorescence was monitored over time using a plate reader (excitation: 380 nm, emission: 460 nm). Since no fluorescence increase over time was observed, the fluorescence at 0 min was plotted as a function of CTSB concentration to generate a calibration curve. The LOD was calculated from the initial slope of the curve using the 3o/m method.

[0385] Determination of CTSB-CLIP Selectivity. To determine the selectivity of CTSB- CLIP, 10 nM CTSB-CLIP was incubated with 20 nM of different proteases and proteins in CTSB Buffer for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). The proteases and proteins used were CTSB, 3CL, MMP7, thrombin (THR). CASP3, and bovine serum albumin (BSA). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final CTSB-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read after an additional 30 min using a plate reader (excitation wavelength of 525 nm and an emission at 566 nm).

[0386] Effect of Nucleases on CLIP Stability. In a low-volume black 384-well plate (Fisher Scientific, Coming™ 384-Well Solid Black or White Polysty rene Microplates, Non-binding, Flat Bottom, Cat No: 09-761-86), 100 nM FQ-CLIP was prepared in IX DNase I reaction buffer provided with the DNase I enzyme (ThermoFisher, DNase I, RNase-free, 1 U/pL, Cat No: EN0521). To each well, 0.1 U, 0.05 U, 0.025 U, 0.01 U, or 0.001 U of DNase I was added to a total reaction volume of 40 pL. A control containing FQ-3CL-CLIP without DNase I was included. The plate was immediately placed in a plate reader, and fluorescence was measured at an excitation wavelength of 648 nm and an emission wavelength of 700 nm.

[0387] Performance of CLIPs in Serum'. 10 nM 3CL-CLIP was incubated with 10% human serum (Millipore Sigma, Human Serum, Cat No: H4522-20ML), with and without 1 pM 3CL protease, for 30 minutes at 25°C with shaking at 1500 RPM (Benchmark Multi-Therm Heating Shaker, Cat No: H5000-H). After incubation, the samples were diluted 10-fold with CRISPR Buffer containing RNP (final concentrations: 10 nM Casl2a, 10 nM gRNA, and 2.5 pL DNase Alert per 100 pL solution). The final concentrations during fluorescence measurement in the CRISPR assay were 1 nM for 3CL-CLIP and 1% for serum. A control containing 1% serum with RNP was included. For comparison, samples of 3CL-CLIP without serum, with and without 1 pM 3CL protease, were processed concurrently. The fluorescence from the CRISPR assay was read using a plate reader after an additional 15 min (excitation wav elength of 525 nm and an emission at 566 nm).

[0388] For comparison, 1 pM 3CL-CFP was incubated with human serum (Millipore Sigma, Human Serum, Cat No: H4522-20ML). with and without 1 pM 3CL protease, for 30 minutes at 25°C with shaking at 1500 RPM (Benchmark Multi-Therm Heating Shaker, CatNo: H5000-H). After incubation, samples were diluted 10-fold with CRISPR Buffer, resulting in final concentrations of 100 nM 3CL-CFP and 1% serum during fluorescence measurement in the CRISPR assay. A control containing 1% serum in 90% CRISPR Buffer and 10% 3CL Buffer was also prepared. For comparison, samples of 3CL-CFP without serum, with and without 1 pM 3CL protease, were processed concurrently. The fluorescence from 3CL-CFP was read using a plate reader after an additional 15 min (excitation wavelength of 485 nm and an emission at 535 nm).

[0389] C ell Culture and Sample Preparation'. The primary cell lines used in this study were

NCI-H508 (CCL-253), RKO (CRL-2577), HT29 (HTB-38), and SW-620 (CCL-227). Every primary cell line was purchased from the American Type Culture Collection (ATTC) and cultivated in RPMI-1640, fortified with 10% fetal bovine serum. 1% penicillin/streptomycin, 1% Glutamax, 1% non-essential amino acids (sourced from Gibco), and lOmM HEPES, and kept in an environment-controlled incubator at 37°C with 5% CO2.

[0390] 2,000,000 viable cells were seeded into 6 cm tissue culture plates and left to incubate overnight. For intracellular protease analysis, the cells were first rinsed with PBS (provided by Gibco) to remove extracellular proteases, then physically dislodged via cell scraping into 1 mL PBS. This solution was then lysed via sonication. The sample was alternately placed in a bath sonicator for 5 s and on ice for 5 s for a total of 5 times. The cells were next collected into a pellet by centrifuging the solution at 16.2x g at a temperature of 4°C for a duration of 15 min. The supernatant was collected for analysis.

[0391] Detection of Active Proteases in Cell lysates'.

[0392] Detection of 3CL Protease in 3CL-Spiked Cell Lysates Using 3CL-CLIPs: To determine the ability of CLIPs to detect a specific protease of interest in complex media, NCI- 14508 lysates were spiked with 3CL protease and treated with 3CL-CLIPs. The presence of SARS CoV-2 proteases in these samples is not expected. Therefore, the fluorescence from 3CL-CLIP in the absence of the 3CL protease serves as an estimate of the background in these systems.

[0393] 10 nM of 3CL-CLI was incubated with 15 pL of the cell lysates (~2 million cells/mL) in a total volume of 45 pL of 3CL Buffer with or without 1 pM 3CL protease. The samples were then incubated for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA. and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final 3CL-CLIP concentration while reading fluorescence in the CRISPR assay w as 1 nM. The fluorescence from the CRISPR assay was read using a plate reader after an additional 30 min (excitation wavelength of 525 nm and an emission at 566 nm). Therefore, total assay time is 60 min.

[0394] Detection of CTSB Protease in Cell Lysates Using CTSB-CLIP: To detect active proteases in complex media, lysates derived from different colon cancer cell lines (NCI-H508, SW-620, RKO, and HT29) were treated with CLIPs. In these cell samples. CTSB is expected to have high activity levels based on previous studies [60.67,73.74], Therefore, fluorescence from CTSB-CLIP will be correlated to the activity of CTSB protease. On the other hand, the presence of SARS CoV-2 proteases in these samples is not expected. Therefore, the fluorescence from 3CL-CLIP will serve as an estimate of the background in these systems.

[0395] 10 nM of CLIPs were incubated with 15 pL of the cell lysates (~2 million cells/mL) in a total volume of 45 pL of IX PBS. The samples w ere then incubated for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final CTSB-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read using a plate reader after an additional 30 min (excitation wavelength of 525 nm and an emission at 566 nm). Therefore, total assay time is 60 min.

[0396] Additional experiments were performed using 5 pL, 3 pL. and lower volumes of the cell lysates. However, no signal was observed for samples containing <3 pL lysates.

[0397] Detection of CTSB Protease in Cell Lysates with Commercial CTSB Reporter: 1 pM of CTSB-CFP was incubated with 15 pL of various cell lysates (~2 million cells/mL) in a total volume of 45 pL of IX PBS. The samples were then incubated for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer. Therefore, the final CTSB-CFP concentration at fluorescence reading was 100 nM. The fluorescence from CTSB- CFP read using a plate reader after an additional 30 min (excitation wavelength of 380 nm and an emission at 460 nm). Therefore, total assay time is 60 min.

[0398] Effect of CTSB Inhibitor on Fluorescence Signal from CTSB-CLIP and CTSB- CFP: To further confirm that the fluorescence signal observed from CTSB-CLIP and CTSB- CFP in the presence of cell lysates stems from the activity of CTSB protease, the study performed additional experiments with a well-known CTSB inhibitor (Sigma Aldrich Cathepsin Inhibitor II, Cat. No. 219385-1MG). It was reasoned that the presence of this inhibitor would lead to a reduction in the fluorescence signal, thereby confirming its dependency on CTSB protease activity.

[0399] 10 nM of CTSB-CLIP was incubated with 6 pL of RKO cell lysates (~2 million cells/mL) in a total volume of 45 pL of IX S with or without 667 pM CTS inhibitor. The samples were then incubated for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker, Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a, 10 nM by gRNA, and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final CTSB-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read using a plate reader after an additional 60 min (excitation wavelength of 525 nm and an emission at 566 nm). Therefore, total assay time is 90 min.

[0400] 1 pM of CTSB-CFP was incubated with 6 pL of RKO cell lysates (~2 million cells/mL) in a total volume of 45 pL of IX S with or without 667 pM CTS inhibitor. The samples were then incubated for 30 min with shaking (Benchmark, Multi-Therm Heating Shaker. Cat No: H5000-H) at 1500 RPM at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer. Therefore, the final CTSB-CFP concentration while reading fluorescence in the CRISPR assay for 100 nM. The fluorescence from CTSB-CFP read using a plate reader after an additional 60 min (excitation wavelength of 380 nm and an emission at 460 nm). Therefore, total assay time is 90 min.

[0401] Data Analysis: All data reported is the average of 3 replicate measurements unless otherwise mentioned. Error bars represent the standard deviation of these measurements.

[0402] Normalized fluorescence is calculated by normalizing the highest fluorescence value in a data set to 100%. Normalized fluorescence is used for FIGS. 2A-2D and FIGS. 3A- 3B

[0403] Fluorescence enhancement is defined as ratio of the fluorescence values of a sample (If) to the fluorescence of the RNP complex (1_RNP) or buffer Ibuffer) at a given timepoint. During error propagation, error from the denominator is ignored. Fluorescence enhancement was calculated using Equation 4:

[0404] Relative fluorescence is defined as the ratio of the fluorescence values of a sample treated with protease ( l₊p_rotease ) ^t0 the fluorescence values the sample without protease (1-Protease ) ^al the same time point. During error propagation, error from the denominator is ignored. Relative fluorescence was calculated using Equation 5:

[0405] Corrected normalized fluorescence is used in melting temperature analysis shown in FIG. 2B. In this experiment, fluorescence of FQ-3CL-CLIP is expected to increase with rising temperature due to enhanced separation between the fluorophore and the quencher.

However, since fluorescence itself can vary with temperature, the study conducted control experiments using only Dye-labeled Blocking DNA to adjust for this temperature dependence. The fluorescence readings from the Dye-labeled Blocking DNA at various temperatures ^Dye-iabeied Blocking DNA, T) were used to correct for the fluorescence values in other samples

(fsampie.r)- The temperature-corrected fluorescence value (Icorrected.r) is given by Equation 6:

[0406] Subsequently, the maximum fluorescence value observed across the data set was assigned a value of 100, while the minimum was set to 0 to correct for background noise. All other fluorescence readings were scaled between these two extremes accordingly. Corrected normalized fluorescence was calculated using Equation 7: . « « 100 _z eq. 7)

[0407] Relative abundance (%) is used for showing mass spectrometry data in FIG. 2A. It is calculated by setting the highest m/z value to 100%.

Additional Results and Discussion

[0408] Tm Analysis: To determine what proportion of the initiator strand must be hybridized to the blocking strand so that the CLIP remains in a hairpin-like structure at room temperature (25°C), the study performed Tm analysis using IDT OligoAnalyzerTM on hairpins comprised entirely of DNA. To closely mimic the structure of CLIPs. the non-DNA portion of the CLIP was replaced with DNA of comparable length. The contour length of amino acids ranges from 0.36 nm to 0.4 nm, the PEG linker's length is approximately 1.4 nm [75], and the length of ssDNA is reported to be between 0.3 nm to 0.6 nm per base [76-78], Therefore, the non-DNA portion of the CLIP (which can be betw een 6.4-6.8 nm in length if the CLIP has 10 aa) can be approximated using ssDNA containing 10-30 bases.

[0409] The study modeled the T_m of DNA hairpins of the form InXwBn and InX^Bn. In represents the initiator strand containing 24 bases. X represents ssDNA bases used to mimic the non-DNA portion of the CLIP. B_n represents the blocking strand containing n bases (n=2~ 13). Hairpins of the form 724X10/L have a stem length of n base pairs and a loop size of 24-/7+10. Hairpins of the form BdX Bn have a stem length of n base pairs and a loop size of 24-/7+30. The loop size decreases as the number of bases in the blocking strand increases.

[0410] After proteolytic cleavage, CLIPs are expected to form an intermolecular duplex with lower T_m compared to the hairpin-like structure. To estimate the T_m of the DNA duplex formed after proteolysis, the study also computed the T_m of dsDNA with n bases. The sequence of these bases is identical to the sequence in the stem portion of the hairpins. All calculated T_m are shown in degree Celsius in TABLE 4, TABLE 5, TABLE 6, and TABLE 11.

[0411] The results indicate that CLIPs with 5-9 base pairs in the stem would maintain hairpin-like structures under the different assay conditions used (DNA concentration 1-10 nM; 100-300 mM Na⁺; 20-40 mM Mg²⁺) but would dissociate upon proteolytic cleavage. The study chose 8 base pairs as the optimal stem length. This range encompasses what is typically understood as physiological ionic strength (-150 mM).

[0412] Structures of DNA hairpins of the form h XsoBs and IUXWBS are shown in FIGS.

6A-6E

[0413] MALDLMS Data: TABLE 9 shows MALDI-MS data of 3CL, CASP3, CTSB, and MMP7-CLIPs and intermediates. TABLE 10 shows MALDLMS data of fluorophore quencher CLIP.

[0414] UV-Vis Characterization of CLIPs: First. peptide-DNA conjugates were synthesized through copper free click chemistry using azido-peptides and dibenzocyclooctyne (DBCO) modified DNA. Initiator DNA was treated with increasing concentrations of azide pep-3CL for 24 h and the reaction progression was observed though monitoring the DBCO’s characteristic 310 nm absorbance. Treatment of 0: 1. 1: 1, 3: 1, and 5: 1, azide to DBCO was measured. In samples of 3 : 1 and 5 : 1 equivalents the 310 nm peak appears to have disappeared fully indicating a complete conjugation (FIGS. 25A-25B). Therefore, all DBCO azide click chemistry used at least a 3:1 ratio to ensure complete product conversion unless otherwise noted.

[0415] Next, an azido PEG linker was installed on the peptide-DNA conjugate. The final CLIP structure (i.e. the DNA-peptide-DNA triblock) was synthesized by reacting linker- peptide-DNAs with Blocking DNA using copper free click chemistry. UV-Vis spectroscopy was used to confirm attachment of Blocking DNA to peptide-DNA linkers. 5: 1 molar ratio was used with respect to DBCO Blocking DNA to peptide-DNA linkers. Therefore, with 100% conjugation a 20% absorbance decrease at 310 nm is theoretically expected. Upon treatment the DBCO’s characteristic absorbance at 310 nm decreases approx. 18%, indicating that the azide on the peptide-DNA linker has successfully conjugated (FIG. 26).

[0416] Gel Electrophoresis Characterization of CLIPs: FIGS. 27A-27C show gel electrophoresis characterization.

[0417] CRISPR-Cas 12a Assays with 3CL-CLIP:

[0418] Optimizing CRISPR-Casl2a Assays'. The study first determined the optimal conditions for CRISPR-Casl2a assays. Specifically, the study investigated how the concentration of Initiator DNA (FIG. 28) and the RNP complex (FIG. 11B) affect the fluorescence signal observed.

[0419] Next, the study verified that incubating the Initiator DNA alone with proteases does not impact the CRISPR signal (FIG. 11C). Following this, the study performed CRISPR assays with 3CL-CLIPs in the presence and absence of proteases. It is important to note that 3CL- CLIPs must be purified from any free Initiator DNA or else background signal is observed (FIGS. 16A-16B). Moreover, the fluorescence signal is affected by the concentration of 3CL- CLIP used in the CRISPR assay (FIGS. 16C-16F).

[0420] Based on these results, it was identified that 1 nM 3CL-CLIP and 10 nM RNP are optimal for these assays. Under these conditions, the limit of detection of 3CL-CLIP is 115 nM (FIGS. 161- 16J)

[0421] Effect of Initiator DNA Concentration on CRISPR-Cas 12a Assays: To evaluate which concentration of DNA is most optimal for CRISPR-Cas 12a assays, RNP was held at a constant 10 nM was treated with increasing concentrations of Initiator DNA. It was observed that an increased concentration of Initiator DNA correlates to an increased CRISPR-Cas 12a fluorescent response. Concentrations above 1 nM show larger standard deviation in triplicate and odd trends while concentrations at or below 0.1 nM show minimal signal. To balance both a need for high signal, high repeatability, and to use fewer reagents a concentration of 1 nM Initiator DNA was chosen as optimal.

[0422] Effect of Increasing RNP Concentration on CRISPR-Cas 12a Assays: To discover an optimal RNP concentration for CRISPR assays, a fixed concentration of Initiator DNA (1 nM) was treated with RNP at concentrations of 100 nM, 10 nM. 1 nM and 0.1 nM. These samples were then subjected to CRISPR assays. Concentrations of RNP below 1 nM show incomplete saturation within 60 min while concentrations around 10 nM saturate within the time frame. An RNP concentration of 10 nM was chosen due to its ability to saturate within 1 h with 1 nM of DNA activator. [0423] Effect on Protease Incubation on the Ability of Initiator DNA to Activate CRISPR- Casl2a Signaling'. To investigate whether incubation of the Initiator DNA with proteases affect CRIPSR assays, 10 nM of Initiator DNA was incubated with 3 pM 3CL protease for 2 h and then subjected to a CRISPR assay. In doing so there was no change seen with the activation of the Initiator DNA with and without protease incubation. This implies that 3CL protease does not interfere with the binding and activation of Initiator DNA with RNP complexes.

[0424] Effect of Purification of 3CL-CLIP on CRISPR-Casl2a Response. Impure and HPLC purified samples at 10 nM 3CL-CLIPs were incubated with 3 pM 3CL protease for 2 h and diluted 10-fold with CRISPR buffer before running a CRISPR assay. The final probe concentration in the CRISPR assay was 1 nM. Impure CLIPs show high background, likely due to the presence of non-hairpining unreacted species, while after HPLC purification, CLIP mixtures show retention of the same signal seen in impure samples but with the decrease of signal in unincubated samples.

[0425] Effect of CLIP Concentration on CRISPR-Cas 12a Assays: 3CL-CLIPs were serially diluted by 10-fold from 10 nM to 10 pM. incubated with 3 pM of 3 CL protease for 2 h, and then subjected to the CRISPR-Casl2a fluorescence assay. Minimal increase in fluorescence is observed using CLIP concentrations below 0.1 nM, whereas 10 nM CLIPs begin to show background signal in the absence of protease. Therefore, an optimal concentration of 1 nM CLIPs (in the final CRISPR buffer) was used for subsequent experiments.

[0426] Limit of Detection of 3CL-CLIPs: To determine the limit of detection (LOD) for the 3CL-CLIP, 10 nM 3CL-CLIP was incubated with varying concentrations of 3CL protease (500 nM, 250 nM, 125 nM, and 0 nM) in 3CL Buffer for 30 min with at RT (at 25°C). Following this, the samples were diluted 10-fold with CRISPR Buffer containing the RNP (final concentration of 10 nM by Casl2a. 10 nM by gRNA. and 2.5 uL/100 uL total solution of DNase Alert). Therefore, the final 3CL-CLIP concentration while reading fluorescence in the CRISPR assay was 1 nM. The fluorescence from the CRISPR assay was read over time. The fluorescence at the 30 min time point from the CRISPR assay was plotted as a function of 3CL concentration to form a calibration curve. From the initial slope of this calibration curve, the LOD was calculated using the 3o/m method. The LOD was found to be 116 nM.

[0427] Comparison of CLIP Hairpins with DNA-only Hairpins: CLIPs were designed with 8 base pairs in the stem. As mentioned previously, this length was chosen based on T_m considerations. To compare the function of CLIPs with DNA-only hairpins of similar lengths, the study performed CRISPR assays using hairpins made entirely of DNA. The study also included controls with the Initiator DNA hybridized to Blocking DNA of different lengths. The contour length of amino acids ranges from 0.36 nm to 0.4 nm, the PEG linker's length is approximately 1.4 nm [75], and the length of ssDNA is reported to be between 0.3 nm to 0.6 nm per base [76-78], Therefore, the non-DNA portion of the CLIP (which can be between 6.4- 6.8 nm in length if the CLIP has 10 aa) can be approximated using ssDNA containing 10-30 bases.

[0428] The study used two DNA hairpins. These hairpins are of the form IiH' dC and hiTsoBs. hi represents the initiator strand containing 24 bases. Bs represents the blocking strand containing bases. hiTioBs is denoted as DNA Hairpin 10-8 and hiT oBs is denoted as DNA Hairpin 30-8. Both hairpins have a stem length of base pairs. hiTioBs has a loop size of 24 - 8 + 10 = 26 bases. hiT Hh has a loop size of 24 - 8 + 30 = 46 bases.

[0429] The study first examined the impact of different lengths of Blocking DNA on CRISPR signaling when the Initiator and Blocking DNA form intermol ecul ar double strands (FIGS. 11D-11E and FIGS. 12A-12C). These results show that a 24-nt Blocking DNA, fully complementary to the Initiator DNA, completely inhibits CRISPR activation, whereas a 16-nt Blocking DNA, with substantial but incomplete complementarity, does not. This suggests that RNP complexes can bind to partially blocked Initiator DNA through strand displacement when intermolecular double strands are present.

[0430] When DNA Hairpin 10-8 and DNA Hairpin 30-8 were used - both containing 8-bp intramolecular double strands - CRISPR activation was observed (FIG. 29). This indicates that RNP complexes can still engage with partially blocked Initiator DNA via strand displacement, even with intramolecular double strands.

[0431] However, when using CLIPs with intramolecular double strands, no CRISPR activation occurred, despite the Blocking DNA being only 8 bases long. This underscores the unique structural properties of CLIPs that prevent strand displacement by the RNP complex, highlighting their distinctive interaction dynamics compared to DNA-only structures.

[0432] CRISPR Activation with Initiator DNA Hybridized to 24-nt and 16-nt Blocking DNA To investigate how blocking the Initiator DNA impacts CRISPR signal, 1 nM of the single-stranded Initiator DNA strand was treated with 10 nM of the 16-nt Blocking DNA or 24-nt Blocking DNA. The 24-nt Blocking DNA fully inhibited CRISPR signal whereas the 16- nt Blocking DNA did not. These results suggest that RNP cannot access fully double-stranded sequences, while, with partial complements, CRISPR-Casl2a can still access the Initiator DNA though toehold-mediated strand displacement.

[0433] CRISPR Activation with DNA-only Hairpins : CRISPR assays were conducted using

1 nM concentration of DNA Hairpin 10-8 and DNA Hairpin 30-8, both featuring 8-base pair intramolecular hairpins. These hairpins were capable of activating CRISPR, producing fluorescence levels comparable to those from Initiator DNA alone. This demonstrates that the presence of an intramolecular double strand in these hairpins does not hinder CRISPR signaling. In contrast, CLIPs, which also possess an 8-base pair DNA stem, do not activate CRISPR. This difference suggests that the peptide and linker elements incorporated into the CLIPs' loops render them inaccessible to the RNP complex, highlighting a significant structural impact on their functionality.

[0434] CRISPR assays using DNA Hairpin 30-8 revealed significant insights when Blocking DNA of various lengths were added to the hairpin. Introducing 24-nt Blocking DNA, which is fully complementary to the hairpin's Initiator DNA sequence, effectively inactivates CRISPR (FIGS. 13A-13B). This inactivation occurs because the 24-nt Blocking DNA opens the hairpin through toehold-mediated strand displacement (FIGS. 30A-30C).

[0435] Conversely, adding 16-nt Blocking DNA, which covers only part of the Initiator DNA sequence and not the hairpin stem, initially diminishes the CRISPR signal. Over time, however, the signal increases, resembling a positive cooperativity curve (FIGS. 13A-13B). This observation could be attributed to factors such as DNA that is misfolded and not in hairpin form or slow disruption of the hairpin stem by CRISPR-Casl2a due to strand displacement. Once initial activation occurs, the CRISPR-Casl2a enzymes, capable of cleaving ssDNA, begin to degrade the hairpin's loop region. This degradation releases new Initiator DNA, leading to an amplification cycle (FIGS. 30A-30C).

[0436] In contrast, the loop structure of CLIPs comprises both peptide and DNA, with the DNA portion being part of the Initiator DNA sequence. If CRISPR-Casl2a cleaves this DNA within the loop, the full Initiator DNA sequence is not intact, preventing further activation of CRISPR-Casl2a (FIG. 15). This structural feature makes CLIPs less susceptible to the amplification cycle typical in DNA-only hairpins, underscoring a significant difference in how CLIPs interact with CRISPR-Casl2a compared to standard DNA hairpins.

[0437] Comparison of 3CL-CLIP with Fluorogenic Substrates: To benchmark the ability of CLIPs to detect active proteases, a commercial 3CL protease reporter (3CL-CFP) was employed in this study. 3CL-CFPs were also used to confirm that the purchased proteases are active. Data from these experiments are shown in the main text.

[0438] To assess the impact of DNA on enhancing the fluorescence signal, the study synthesized a CLIP equipped with a fluorophore (Cy5) and a quencher (BHQ) at opposite ends, designated as FQ-3CL-CLIP. This construct was purified via gel electrophoresis (FIG. 9B) and characterized using UV-Vis spectroscopy (FIG. 31). The FQ-3CL-CLIP serves as a fluorogenic reporter for the presence of active proteases, as proteolytic cleavage between the fluorophore and the quencher will lead to an increase in fluorescence (FIG. 10 and FIG. 32). [0439] UV-Vis spectroscopy was used to support successful synthesis of FQ-3CL-CLIP Probes. A UV-Vis spectrum confirms the presence of DNA, BHQ, and cyanine-5 due to their characteristic absorbances at 260 nm, -580 nm, -650 nm respectively. The absence of DBCO’s characteristic 310 nm peak indicates successful conjugation of quencher-DNA linker species and dye labeled blocking strands.

[0440] Protease-mediated degradation of FQ-3CL-CLIP results in the separation of the fluorophore and the quencher and generates a fluorescence signal. This probe serves as an additional control to support the intended DNA disassociation of CLIPs following proteolytic digestion.

[0441] When comparing the fluorescence from the FQ-3CL-CLIP assay with that from a CRISPR assay using 3CL-CLIP, the CRISPR-enhanced 3CL-CLIP exhibited a stronger signal than the FQ-3CL-CLIP, even at concentrations 100 times lower (FIG. 33). This indicates that DNA effectively amplifies the signal in these assays.

[0442] Comparison of CTSB-CLIP with Commercial CTSB Probes: These results show that CTSB-CLIP, at a final concentration of 1 nM, can detect sub-nanomolar concentrations of the protease CTSB. In contrast, a commercial fluorogenic probe for CTSB (CTSB-CFP) failed to detect similar levels of proteases under comparable conditions.

[0443] CTSB-CFP and CTSB-CLIP (10 nM) were incubated with 10 nM CTSB protease for 30 min in CTSB buffer. Following this, the samples were diluted 10-fold using CRISPR buffer to a final concentration of 1 nM by probe. In case of CTSB-CLIP, the CRISPR buffer was supplemented with CRISPR-Casl2a RNP and DNase Alert reporters. Fluorescence readings were taken after an additional 120 min.

[0444] To determine the limit of detection (LOD) for CTSB-CFP, 1 pM CTSB-CFP was incubated with varying concentrations of CTSB protease (25 nM, 10 nM, 5 nM, 0. 1 nM, and 0 nM) in CTSB Buffer for 30 min at 25°C. After incubation, the samples were diluted 10-fold with CRISPR Buffer, resulting in a final CTSB-CFP concentration of 100 nM during fluorescence measurement in the CRISPR assay. Fluorescence values were plotted as a function of CTSB concentration to generate a calibration curve. The LOD was calculated from the initial slope of this curve using the 3o/m method and determined to be 3.4 nM.

[0445] Performance of CLIPs in Complex Media:

[0446] Stability of CLIPs in the Presence of Nucleases'. DNase I was used to assess the stability of CLIP structures against nuclease degradation. DNase I, which digests both single-

I l l and double-stranded DNA, was applied to FQ-3CL-CLIP at varying concentrations. Upon digestion, the fluorophore and quencher separate, generating a fluorescence signal indicative of probe degradation. These results show that after 30 minutes of treatment, while the CLIP structure is degraded at high DNase I concentrations, it remains stable at DNase activity of 25 U/L or lower, at least up to 30 min. This is more than double the activity of DNase I in human serum (~10 U/L), demonstrating the robustness of the CLIP structure under relevant scenarios [66],

[0447] Stability of CLIPs in Cell Lysates'. It is worth noting that GelRed preferentially stains longer DNA sequences, which explains the presence of only one prominent band in the GelRed channel (the intact CLIP). In the Cy5 channel, faint, faster-migrating bands emerge over time, suggesting progressive degradation of FQ-3CL-CLIP in the cellular lysates. The intact CLIP structure contains both Cy5 and its quencher, BHQ. As the CLIP degrades, Cy5 and BHQ separate, causing an increase in Cy5 fluorescence relative to the intact structure. This artificially enhances the intensity of the fragment bands in the Cy5 channel, making the proportion of fragments appear higher than it actually is. Furthermore, the relatively unchanged intensity of the parent CLIP band in both the GelRed and Cy5 channels indicates that only a small fraction of the CLIP is degraded. Therefore, the CLIP structure is largely stable in cell lysates at least up to 6 h.

[0448] Performance of CLIPs in Serum'. 3CL-CLIP and 3CL-CFP were treated with 3CL protease in the 3CL protease buffer with the addition of 10% human serum. Upon incubation of the probes with serum, it was observed that even without the treatment of the protease there is an increase of background signal of -200% for both the CLIP and CFP. This indicates that there is a significant background due to perhaps non-specific cleavage of the probe's peptide recognition sequence (which is shared with the CLIP and CFP) by a component in the serum. Upon incubating the probes with 3 CL protease, fluorescence increases were observed in both indicating they are working as intended to detect the proteases in the serum.

[0449] Performance of CLIPs in Cell Lysates'. FIGS. 5A-5D shows detection of CTSB using CLIPs in cancer cell lysates.

[0450] Limit of Detection of CTSB Protease Using CTSB-CLIP in SW-620 Lysates'. FIGS. 39A-39B shows the response of CTSB-CLIP to varying amounts of SW-620 colon cancer cell lysates.

[0451] Detection of CTSB in Cell Lysates via Commercial CTSB Probe. FIG. 40 shows fluorescence enhancement of CTSB-CFP after incubation with lysates of HT29, NCI-H508, RKO, and SW-620 cell lines. Conclusion

[0452] In conclusion, CLIPs represent a powerful new class of activity-based sensors, offering a simple, rapid, room temperature method to detect proteases. They outperform commercial probes even at 100 times lower concentrations due to the incorporation of DNA, which serves as a handle for signal amplification. The DNA handle not only enhances detection capabilities beyond those of previous peptide beacons [71,72] but also simplifies the detection process relative to nanoparticle-based approaches. Unlike earlier nucleic acid-based strategies. CLIPs do not require complex synthetic biology or separation of "free" DNA from intact probes, making the detection one-pot and rapid, with overall assay times as fast as 35 minutes (30 min protease incubation followed by 5 min CRISPR assay). CLIPs also eliminate the need for sophisticated equipment by utilizing commonly available plate readers for readouts. Moreover, adjustments to the initiator and blocking strands could optimize CLIP performance in various conditions, such as at elevated temperatures or different ionic strengths. The modular nature of the probe allow s for detecting a variety of proteases in complex matrices simply bychanging the peptide sequence. Importantly, the unique structure of CLIPs ensures that they only activate CRISPR in the presence of a target, providing a solid foundation for detecting not only proteases but also potentially other disease-relevant enzymes with cleavable substrates. While CRISPR-mediated signaling is currently employed to detect the activated initiator strand, integration of other DNA detection techniques could further boost sensitivity, potentially achieving detection at the single-cell level. Taken together, the flexibility, sensitivity, and specificity of CLIPs establish them as a versatile and promising approach for activity -based sensing, making them a valuable addition to the MB toolbox.

EXAMPLE ASPECTS

[0453] Example 1: A composition comprising a nucleic acid-locked beacon, wherein said nucleic acid-locked beacon comprises: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence.

[0454] Example 2: The composition of any examples herein, particularly Example 1, wherein the cleavable domain is recognized and is cleavable by an enzyme of interest.

[0455] Example 3: The composition of any examples herein, particularly Example 2, wherein the enzy me of interest is a protease of interest, and w herein said protease of interest is MMP7, Caspase 3, 3 -chymotrypsin-like main protease (3CL) of SARS-CoV-2, or Cathepsin B. [0456] Example 4: The composition of any examples herein, particularly Examples 1-3, wherein the cleavable domain is a peptide domain, and wherein said peptide domain is from 3 to 75 or more amino acids in length.

[0457] Example 5: The composition of any examples herein, particularly Examples 1-4, wherein the cleavable domain is a peptide domain comprising 80% similarity or more to anyone of SEQ ID NOs: 1-4.

[0458] Example 6: The composition of any examples herein, particularly Examples 1-5. wherein the cleavable domain is a peptide domain comprising 90% similarity or more to any one of SEQ ID NOs: 1-4.

[0459] Example 7: The composition of any examples herein, particularly Examples 1-6, wherein the cleavable domain is a peptide domain comprising any one of SEQ ID NOs: 1-4.

[0460] Example 8: The composition of any examples herein, particularly Examples 1-7, wherein the signaling nucleic acid sequence is configured to produce a detectable signal.

[0461] Example 9: The composition of any examples herein, particularly Example 8, wherein the detectable signal is a fluorescence signal.

[0462] Example 10: The composition of any examples herein, particularly Example 9. wherein the signaling nucleic acid sequence activates a Cas molecule, wherein the Cas molecule cleaves a nucleic acid reporter, thereby causing the fluorescence signal.

[0463] Example 11: The composition of any examples herein, particularly Example 10, wherein the Cas molecule comprises a Cas 12a or Cas 13a RNP system.

[0464] Example 12: The composition of any examples herein, particularly Examples 1- 11, wherein the signaling nucleic acid sequence further comprises a barcode nucleic acid sequence and/or a primer site for amplifying said barcode nucleic acid sequence, and wherein the detectable signal is said amplified barcode nucleic acid sequence.

[0465] Example 13: The composition of any examples herein, particularly Example 12, wherein the barcode nucleic acid sequence is amplified by polymerase chain reaction (PCR), exponential amplification reaction (EXPAR), loop-mediated isothermal amplification (LAMP), next-generation sequencing (NGS), strand displacement amplification (SDA), or hybridization chain reaction.

[0466] Example 14: The composition of any examples herein, particularly Examples 1-

13, wherein the signaling nucleic acid sequence is from 4 to 100 nucleotides in length.

[0467] Example 15: The composition of any examples herein, particularly Examples 1-

14, wherein the signaling nucleic acid sequence comprises DNA, RNA, or modified oligonucleotides. [0468] Example 16: The composition of any examples herein, particularly Example 15, wherein the signaling nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 6, 8, or 10.

[0469] Example 17: The composition of any examples herein, particularly Examples 15-

16, wherein the signaling nucleic acid sequence comprises 90% similarity⁷ or more to any one of SEQ ID NOs: 6. 8, or 10.

[0470] Example 18: The composition of any examples herein, particularly Examples 15-

17, wherein the signaling nucleic acid sequence comprises any one of SEQ ID NOs: 6, 8, or 10.

[0471] Example 19: The composition of any examples herein, particularly Examples 1-

18, wherein the blocking nucleic acid is from 4 to 20 nucleotides in length.

[0472] Example 20: The composition of any examples herein, particularly Examples 1-

19, wherein the blocking nucleic acid sequence comprises DNA, RNA, or modified nucleotides.

[0473] Example 21: The composition of any examples herein, particularly Example 20, wherein the blocking nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 7, 9, or 14-15.

[0474] Example 22: The composition of any examples herein, particularly Examples 20-

21, wherein the blocking nucleic acid sequence comprises 90% similarity or more to any one of SEQ ID NOs: 7. 9, or 14-15.

[0475] Example 23: The composition of any examples herein, particularly Examples 20-

22, wherein the blocking nucleic acid sequence comprises any one of SEQ ID NOs: 7, 9, or 14- 15.

[0476] Example 24: The composition of any examples herein, particularly Examples 1-

23, wherein, when the cleavable domain is intact, the nucleic acid portion of the beacon forms a secondary structure selected from a hairpin structure and a circularized structure.

[0477] Example 25: The composition of any examples herein, particularly Examples 1-

24, wherein, when the cleavable domain is intact, the blocking nucleic acid sequence and the signaling nucleic acid sequence are at least partially hybridized.

[0478] Example 26: The composition of any examples herein, particularly Example 25, wherein, when the cleavable domain is intact, the blocking nucleic acid sequence and the signaling nucleic acid sequence are at least partially hybridized under a given set of conditions, and further wherein, upon cleavage of the cleavable domain by the enzyme, the blocking nucleic acid sequence and the signaling sequence at least partially denature under said given set of conditions.

[0479] Example 27: The composition of any examples herein, particularly Example 26, wherein the given set of conditions comprise temperature, pH, and/or salt concentration.

[0480] Example 28: The composition of any examples herein, particularly Examples 26- 27, wherein at least partial denaturation of the blocking nucleic acid sequence and signaling nucleic acid sequence allows the signaling nucleic acid sequence to produce a detectable signal. [0481] Example 29: The composition of any examples herein, particularly Example 28, wherein at least partial hybridization of the blocking nucleic acid sequence and the signaling nucleic acid sequence at least partially reduces production of the detectable signal by the signaling nucleic acid sequence

[0482] Example 30: The composition of any examples herein, particularly Examples 1- 29, wherein the nucleic acid-locked beacon further comprises a linker.

[0483] Example 31: The composition of any examples herein, particularly Example 30, wherein the linker is between the cleavable domain and the blocking nucleic acid.

[0484] Example 32: The composition of any examples herein, particularly Examples 30- 31 , wherein a portion or all of the linker comprises a bifunctional molecule, such as a PEG- based linker or a nucleic acid based linker.

[0485] Example 33: The composition of any examples herein, particularly Example 1-32, wherein the nucleic acid-locked beacon comprise one or more modifications.

[0486] Example 34: The composition of any examples herein, particularly Example 33, wherein the modifications make the beacon more stable and/or more resistant to degradation.

[0487] Example 35: The composition of any examples herein, particularly Examples 33-

34, wherein the modifications are chemical modifications, including but not limited to non- naturally occurring amino acids or nucleic acids.

[0488] Example 36: The composition of any examples herein, particularly Examples 1-

35, wherein the beacon is barcoded.

[0489] Example 37: The composition of any examples herein, particularly Examples 1-

36, wherein the beacon is associated with a nanostructure such as a nanoparticle or a silica- based microparticle.

[0490] Example 38: The composition of any examples herein, particularly Example 37, wherein the nanoparticle is a gold nanoparticle.

[0491] Example 39: A composition comprising two or more nucleic acid-locked beacons, wherein each of said nucleic acid-locked beacon comprises: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein the two or more nucleic acid-locked beacons comprise different peptide sequences.

[0492] Example 40: The composition of any examples herein, particularly Example 39, wherein the two or more nucleic acid-locked beacons allows for the detection of two or more different enzymes in the same assay (multiplex).

[0493] Example 41: A method of detecting an enzyme of interest in a sample, the method comprising: a) exposing the enzyme of interest to a composition comprising a nucleic acid- locked beacon, wherein said nucleic acid-locked beacon comprises: i) a cleavable domain; ii) a signaling nucleic acid sequence: and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and b) detecting the signal produced by the signaling nucleic acid sequence, thereby detecting the enzyme of interest.

[0494] Example 42: The method of any examples herein, particularly Example 41. wherein the enzyme of interest is a marker of cancer, inflammation, infection, virus, cardiovascular disease, or other disease or disorder.

[0495] Example 43: The method of any examples herein, particularly Examples 41-42, wherein the enzyme of interest is a protease of interest, and wherein said protease of interest is MMP7, Caspase 3, 3CL, or Cathepsin B.

[0496] Example 44: The method of any examples herein, particularly Examples 41-43, wherein the cleavable domain comprises 80% similarity or more to any one of SEQ ID NOs: 1-4.

[0497] Example 45: The method of any examples herein, particularly Examples 41-44, wherein the detectable signal is a fluorescence signal.

[0498] Example 46: The method of any examples herein, particularly Example 45, wherein the signaling nucleic acid sequence activates a Cas molecule, wherein the Cas molecule cleaves a nucleic acid reporter, thereby causing the fluorescence signal.

[0499] Example 47: The method of any examples herein, particularly Examples 41-46, wherein the signaling nucleic acid sequence further comprises a barcode nucleic acid sequence and/or a primer site for amplifying said barcode nucleic acid sequence, and wherein the detectable signal is said amplified barcode nucleic acid sequence. [0500] Example 48: The method of any examples herein, particularly Examples 41-47, wherein the signaling nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 6, 8, or 10.

[0501] Example 49: The method of any examples herein, particularly Examples 41-48, wherein the blocking nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 7, 9. or 14-15.

[0502] Example 50: The method of any examples herein, particularly Examples 41-49. wherein, when the cleavable domain is intact, the blocking nucleic acid sequence and the signaling nucleic acid sequence are at least partially hybridized under a given set of conditions, and further wherein, upon cleavage of the cleavable domain, the blocking nucleic acid sequence and the signaling sequence at least partially denature under said given set of conditions.

[0503] Example 51: The method of any examples herein, particularly Example 50, wherein the given set of conditions comprise temperature, pH, and/or salt concentration.

[0504] Example 52: The method of any examples herein, particularly Examples 50-51, wherein at least partial denaturation of the blocking nucleic acid sequence and signaling nucleic acid sequence allows the signaling nucleic acid sequence to produce a detectable signal, and wherein at least partial hybridization of the blocking nucleic acid sequence and the signaling nucleic acid sequence at least partially reduces production of a detectable signal by the signaling nucleic acid sequence.

[0505] Example 53: The method of any examples herein, particularly Examples 41-52, wherein the nucleic acid-locked beacon comprise one or more modifications.

[0506] Example 54: The method of any examples herein, particularly Example 53, wherein the modifications make the beacon more stable and/or more resistant to degradation.

[0507] Example 55: The method of any examples herein, particularly Examples 53-54, wherein the modifications are chemical modifications, including but not limited to non- naturally occurring amino acids or nucleic acids.

[0508] Example 56: The method of any examples herein, particularly Examples 41-55, wherein the beacon is barcoded.

[0509] Example 57: The method of any examples herein, particularly Examples 41-56. wherein the beacon is associated with a nanostructure such as a nanoparticle or a microparticle such as silica beads.

[0510] Example 58: The method of any examples herein, particularly Examples 41-57, wherein the method takes place in vitro, in vivo, or ex vivo. [0511] Example 59: The method of any examples herein, particularly Examples 41-58, wherein steps a) and b) further comprise: a) exposing two or more enzymes of interest to a composition comprising two or more nucleic acid-locked beacons, wherein each nucleic acid- locked beacon comprises: i) a cleavable domain specific to one enzyme of interest; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows each enzyme to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and b) detecting the signals produced by each signaling nucleic acid sequence, thereby detecting each enzy me of interest.

[0512] Example 60: The method of any examples herein, particularly Example 59, wherein the two or more nucleic acid-locked beacons allows for the detection of two or more different enzymes in the same assay (multiplex).

[0513] Example 61: A method of determining usefulness of a test compound in modulating an enzyme, the method comprising: a) exposing the test compound to the enzyme to form a test composition; b) exposing the test composition to a composition comprising a nucleic acid-locked beacon, wherein said nucleic acid-locked peptide comprises: i) a cleavable domain; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and c) detecting the signal produced by the signaling nucleic acid sequence, thereby detecting the enzyme of interest; and d) using said signal to determine an effect of the test compound on action of the enzyme.

[0514] Example 62: The method of any examples herein, particularly Example 61, wherein the effect of the test compound on action of the enzyme comprises an increase in enzy me amount or activity, and wherein the signal is greater than a reference signal produced by the enzyme not exposed to the test compound.

[0515] Example 63: The method of any examples herein, particularly Example 61. wherein the effect of the test compound on action of the enzyme comprises a decrease in enzyme amount or activity⁷, wherein the signal is lesser than a reference signal produced by the enzyme not exposed to the test compound. [0516] Example 64: The method of any examples herein, particularly Examples 61-63, wherein the enzyme of interest is a marker of cancer, inflammation, infection, virus, cardiovascular disease, or other disease or disorder.

[0517] Example 65: The method of any examples herein, particularly Examples 61-64, wherein the enzyme of interest is a protease of interest, and wherein said protease of interest is MMP7, 3CL, Caspase 3. or Cathepsin B.

[0518] Example 66: The method of any examples herein, particularly Examples 61-65. wherein the cleavable domain is a peptide domain comprising 80% similarity or more to any one of SEQ ID NOs: 1-4.

[0519] Example 67: The method of any examples herein, particularly Examples 61-66, wherein the detectable signal is a fluorescence signal.

[0520] Example 68: The method of any examples herein, particularly Example 67, wherein the signaling nucleic acid sequence activates a Cas molecule, wherein the Cas molecule cleaves a nucleic acid reporter, thereby causing the fluorescence signal.

[0521] Example 69: The method of any examples herein, particularly Examples 61-68, wherein the signaling nucleic acid sequence further comprises a barcode nucleic acid sequence and/or a primer site for amplifying said barcode nucleic acid sequence, and wherein the detectable signal is said amplified barcode nucleic acid sequence.

[0522] Example 70: The method of any examples herein, particularly Examples 61-69, wherein the signaling nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 6, 8, or 10.

[0523] Example 71: The method of any examples herein, particularly Examples 61-70, wherein the blocking nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 7, 9. or 14-15.

[0524] Example 72: The method of any examples herein, particularly Examples 61-71, wherein, when the cleavable domain is intact, the blocking nucleic acid sequence and the signaling nucleic acid sequence are at least partially hybridized under a given set of conditions, and further wherein, upon cleavage of the cleavable domain, the blocking nucleic acid sequence and the signaling sequence at least partially denature under said given set of conditions.

[0525] Example 73: The method of any examples herein, particularly Example 72, wherein the given set of conditions comprise temperature, pH, and/or salt concentration.

[0526] Example 74: The method of any examples herein, particularly Examples 72-73, wherein at least partial hybridization of the blocking nucleic acid sequence and the signaling nucleic acid sequence at least partially reduces production of a detectable signal by the signaling nucleic acid sequence, and wherein at least partial denaturation of the blocking nucleic acid sequence and signaling nucleic acid sequence allows the signaling nucleic acid sequence to produce a detectable signal.

[0527] Example 75: The method of any examples herein, particularly Examples 61-74, wherein the nucleic acid-locked beacon comprise one or more modifications.

[0528] Example 76: The method of any examples herein, particularly Example 75, wherein the modifications make the beacon more stable and/or more resistant to degradation.

[0529] Example 77: The method of any examples herein, particularly Examples 75-76, wherein the modifications are chemical modifications, including but not limited to non- naturally occurring amino acids or nucleic acids.

[0530] Example 78: The method of any examples herein, particularly Examples 61-77, wherein the beacon is barcoded.

[0531] Example 79: The method of any examples herein, particularly Examples 61-78, wherein the beacon is associated with a nanostructure such as a nanoparticle or a microparticle such as silica beads.

[0532] Example 80: The method of any examples herein, particularly Examples 61-79. wherein the method takes place in vitro. in vivo. or ex vivo.

[0533] Example 81: The method of any examples herein, particularly Examples 61-80, wherein steps a) and b) further comprise: a) exposing the test compound to two or more enzymes of interest to form a test composition; b) exposing the test composition to a composition comprising two or more nucleic acid-locked beacons, wherein each nucleic acid- locked beacon comprises: i) a cleavable domain specific to one enzyme of interest; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary’ to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows each enzyme to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and c) detecting the signals produced by each signaling nucleic acid sequence, thereby detecting each enzyme of interest; and d) using said signals to determine an effect of the test compound on action of each enzyme.

[0534] Example 82: The method of any examples herein, particularly Example 81, wherein the two or more nucleic acid-locked beacons allows for the detection of two or more different enzy mes in the same assay (multiplex), and wherein the method is used to determine the effect of the test compound on two or more enzymes of interest simultaneously. [0535] Example 83: A composition detected by the method of any examples herein, particularly Examples 61-82.

[0536] Example 84: A diagnostic assay for determining presence of an enzyme, the assay comprising a nucleic acid-locked beacon, wherein said nucleic acid-locked beacon comprises: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence.

[0537] Example 85: The diagnostic assay of any examples herein, particularly Example 84, wherein the assay comprises a composition of any examples herein, particularly Examples 1-40.

[0538] Example 86: A method of treating and/or preventing a disease or disorder, wherein said disease or disorder causes an increase of an enzyme in a subject, the method comprising: a) administering to the subject a composition comprising anucleic acid-locked beacon, wherein said nucleic acid-locked beacon comprises: i) a cleavable domain; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a therapeutic response.

[0539] Example 87: The method of any examples herein, particularly Example 86, wherein the signaling nucleic acid is a single strand anti sense DNA for gene therapy.

[0540] Example 88: The method of any examples herein, particularly Examples 86-87, wherein the enzyme of interest is a marker of cancer, inflammation, infection, virus, cardiovascular disease, or other disease or disorder.

[0541] Example 89: The method of any examples herein, particularly Examples 86-88, wherein the enzyme of interest is a protease of interest, and wherein said protease of interest is MMP7, 3CL, Caspase 3, or Cathepsin B.

[0542] Example 90: The method of any examples herein, particularly Examples 86-89, wherein the cleavable domain is a peptide domain comprising 80% similarity or more to anyone of SEQ ID NOs: 1-4.

[0543] Example 91: The method of any examples herein, particularly Examples 86-90. wherein the signaling nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 6, 8, or 10.

[0544] Example 92: The method of any examples herein, particularly Examples 86-91, wherein the blocking nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 7, 9, or 14-15. [0545] Example 93: The method of any examples herein, particularly Examples 86-92, wherein the nucleic acid-locked beacon comprise one or more modifications.

[0546] Example 94: The method of any examples herein, particularly Example 93, wherein the modifications make the beacon more stable and/or more resistant to degradation.

[0547] Example 95: The method of any examples herein, particularly Examples 93-94, wherein the modifications are chemical modifications, including but not limited to non- naturally occurring amino acids or nucleic acids.

[0548] Example 96: The method of any examples herein, particularly Examples 86-95, wherein the beacon is associated with a nanostructure such as a nanoparticle.

[0549] Example 97: The method of any examples herein, particularly Examples 86-96, wherein step a) further comprises: a) administering to the subject a composition comprising two or more nucleic acid-locked beacon, wherein each nucleic acid-locked beacon comprises: i) a cleavable domain specific to one enzyme; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows each enzyme to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a therapeutic response.

[0550] Example 98: The method of any examples herein, particularly Example 97, wherein the two or more nucleic acid-locked beacons allows for the therapeutic response to two or more different enzymes in the same subject.

[0551] Example 99: A nucleic acid-locked beacon, wherein said nucleic acid-locked beacon comprises: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein the cleavable domain is recognized and is cleavable by an enzyme of interest.

[0552] Example 100: The nucleic acid-locked beacon of any examples herein, particularly Example 99, wherein the enzyme of interest is a marker of cancer, inflammation, infection, virus, cardiovascular disease, or other disease or disorder.

[0553] Example 101: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-100, wherein the enzyme of interest is a protease of interest, and wherein the cleavable domain is a peptide domain.

[0554] Example 102: The nucleic acid-locked beacon of any examples herein, particularly Example 101, wherein said protease of interest is MMP7, Caspase 3, 3-chymotrypsin-like main protease (3CL) of SARS-CoV-2, or Cathepsin B. [0555] Example 103: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-102. wherein the cleavable domain is a peptide domain comprising 80% similarity or more to any one of SEQ ID NOs: 1-4.

[0556] Example 104: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-103, wherein the signaling nucleic acid sequence is configured to produce a detectable signal.

[0557] Example 105: The nucleic acid-locked beacon of any examples herein, particularly Example 104 wherein the signaling nucleic acid sequence activates a Cas molecule, wherein the Cas molecule cleaves a nucleic acid reporter, thereby causing a fluorescence signal.

[0558] Example 106: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-105, wherein the signaling nucleic acid sequence further comprises a barcode nucleic acid sequence and/or a primer site for amplifying said barcode nucleic acid sequence, and wherein the detectable signal is said amplified barcode nucleic acid sequence.

[0559] Example 107: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-106, wherein the signaling nucleic acid sequence is from 4 to 100 nucleotides in length; and wherein the blocking nucleic acid is from 4 to 20 nucleotides in length.

[0560] Example 108: The nucleic acid-locked beacon of any examples herein, particularly Example 107, wherein the signaling nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 6, 8, or 10; and wherein the blocking nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 7, 9. or 14-15.

[0561] Example 109: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-108, wherein, when the cleavable domain is intact: the nucleic acid portion of the beacon forms a secondary structure selected from a hairpin structure and a circularized structure: and the blocking nucleic acid sequence and the signaling nucleic acid sequence are at least partially hybridized.

[0562] Example 110: The nucleic acid-locked beacon of any examples herein, particularly Example 109, wherein, when the cleavable domain is intact, the blocking nucleic acid sequence and the signaling nucleic acid sequence are at least partially hybridized under a given set of conditions, and further wherein, upon cleavage of the cleavable domain by the enzyme, the blocking nucleic acid sequence and the signaling sequence at least partially denature under said given set of conditions; and wherein the given set of conditions comprise temperature, pEI, and/or salt concentration.

[0563] Example 111: The nucleic acid-locked beacon of any examples herein, particularly Example 1 10, wherein at least partial denaturation of the blocking nucleic acid sequence and signaling nucleic acid sequence allows the signaling nucleic acid sequence to produce a detectable signal; and wherein at least partial hybridization of the blocking nucleic acid sequence and the signaling nucleic acid sequence at least partially reduces production of the detectable signal by the signaling nucleic acid sequence.

[0564] Example 112: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-111, wherein the nucleic acid-locked beacon further comprises a linker between the cleavable domain and the blocking nucleic acid.

[0565] Example 113: The nucleic acid-locked beacon of any examples herein, particularly

Example 112, wherein a portion or all of the linker comprises a bifunctional molecule, such as a PEG-based linker or a nucleic acid based linker.

[0566] Example 114: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-1 13, wherein the nucleic acid-locked beacon comprise one or more non-naturally occurring amino acids or nucleic acids; and wherein the non-naturally occurring amino acids or nucleic acids make the beacon more stable and/or more resistant to degradation.

[0567] Example 115: The nucleic acid-locked beacon of any examples herein, particularly Examples 99-114, wherein the beacon is barcoded.

[0568] Example 116: The nucleic acid-locked beacon of any examples herein, particularly

Examples 99-115, wherein the beacon is associated with a nanostructure such as a nanoparticle or a silica-based microparticle.

[0569] Example 117: A composition comprising two or more nucleic acid-locked beacons of any examples herein, particularly Examples 99-1 16, wherein the two or more nucleic acid- locked beacons comprise different peptide sequences; and wherein the two or more nucleic acid-locked beacons allows for detection of two or more different enzy mes in the same assay (multiplex).

[0570] Example 118: A method of detecting an enzyme of interest in a sample, the method comprising: a) exposing the enzyme of interest to the nucleic acid-locked beacon of any examples herein, particularly Examples 99-116, wherein said exposure occurs under conditions which allows the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and b) detecting the signal produced by the signaling nucleic acid sequence, thereby detecting the enzyme of interest.

[0571] Example 119: The method of any examples herein, particularly Example 118, wherein steps a) and b) further comprise: a) exposing two or more enzymes of interest to a composition comprising two or more nucleic acid-locked beacons, wherein each nucleic acid- locked beacon comprises: i) a cleavable domain specific to one enzyme of interest; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows each enzy me to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and b) detecting the signals produced by each signaling nucleic acid sequence, thereby detecting each enzyme of interest; wherein the two or more nucleic acid-locked beacons allows for the detection of two or more different enzy mes in the same assay (multiplex), and wherein the method is used to detect two or more enzy mes of interest simultaneously.

[0572] Example 120: A method of determining usefulness of a test compound in modulating an enzyme, the method comprising: a) exposing the test compound to the enzyme to form a test composition; b) exposing the test composition to the nucleic acid-locked beacon of any examples herein, particularly Examples 99-116, wherein said exposure occurs under conditions which allows the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and c) detecting the signal produced by the signaling nucleic acid sequence, thereby detecting the enzyme of interest; and d) using said signal to determine an effect of the test compound on action of the enzyme.

[0573] Example 121: The method of any examples herein, particularly Example 120, wherein the effect of the test compound on action of the enzyme comprises an increase in enzyme amount or activity⁷, and wherein the signal is greater than a reference signal produced by the enzyme not exposed to the test compound; or wherein the effect of the test compound on action of the enzyme comprises a decrease in enzyme amount or activity, wherein the signal is lesser than a reference signal produced by the enzyme not exposed to the test compound.

[0574] Example 122: The method of any examples herein, particularly Examples 120-121, wherein steps a) and b) further comprise: a) exposing the test compound to two or more enzy mes of interest to form a test composition; b) exposing the test composition to a composition comprising two or more nucleic acid-locked beacons, wherein each nucleic acid- locked beacon comprises: i) a cleavable domain specific to one enzyme of interest; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary' to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows each enzyme to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and c) detecting the signals produced by each signaling nucleic acid sequence, thereby detecting each enzy me of interest; and d) using said signals to determine an effect of the test compound on action of each enzyme; wherein the two or more nucleic acid-locked beacons allows for the detection of two or more different enzymes in the same assay (multiplex), and wherein the method is used to determine the effect of the test compound on two or more enzy mes of interest simultaneously.

[0575] Example 123: A diagnostic assay for determining presence of an enzyme, the assay comprising a nucleic acid-locked beacon, wherein said nucleic acid-locked beacon comprises the nucleic acid-locked beacon of any examples herein, particularly Examples 99-116.

[0576] Example 124: A method of treating and/or preventing a disease or disorder, wherein said disease or disorder causes an increase of an enzyme in a subject, the method comprising: a) administering to the subject a composition comprising the nucleic acid-locked beacon of any examples herein, particularly Examples 99-116, wherein said exposure occurs under conditions which allows the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a therapeutic response.

[0577] Example 125: The method of any examples herein, particularly Example 124. wherein the signaling nucleic acid is a single strand anti sense DNA for gene therapy.

[0578] Example 126: The method of any examples herein, particularly Examples 124-125, wherein step a) further comprises: a) administering to the subject a composition comprising two or more nucleic acid-locked beacon, wherein each nucleic acid-locked beacon comprises: i) a cleavable domain specific to one enzyme; ii) a signaling nucleic acid sequence; and iii) a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein said exposure occurs under conditions which allows each enzy me to recognize and cleave each cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a therapeutic response; and wherein the two or more nucleic acid-locked beacons allows for the therapeutic response to two or more different enzymes in the same subject.

[0579] 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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Claims

1. A nucleic acid-locked beacon, wherein said nucleic acid-locked beacon comprises: a cleavable domain; a signaling nucleic acid sequence; and a blocking nucleic acid sequence at least partially complementary to the signaling nucleic acid sequence; wherein the cleavable domain is recognized and is cleavable by an enzyme of interest.

2. The nucleic acid-locked beacon of claim 1, wherein the enzyme of interest is a marker of cancer, inflammation, infection, virus, cardiovascular disease, or other disease or disorder.

3. The nucleic acid-locked beacon of any one of claims 1-2, wherein the enzy me of interest is a protease of interest, and wherein the cleavable domain is a peptide domain.

4. The nucleic acid-locked beacon of claim 3, wherein said protease of interest is MMP7, Caspase 3, 3-chymotrypsin-like main protease (3CL) of SARS-CoV-2, or Cathepsin B.

5. The nucleic acid-locked beacon of any one of claims 1-4, wherein the cleavable domain is a peptide domain comprising 80% similarity or more to any one of SEQ ID NOs: 1-4.

6. The nucleic acid-locked beacon of any one of claims 1-5, wherein the signaling nucleic acid sequence is configured to produce a detectable signal.

7. The nucleic acid-locked beacon of claim 6, w herein the signaling nucleic acid sequence activates a Cas molecule, wherein the Cas molecule cleaves a nucleic acid reporter, thereby causing a fluorescence signal.

8. The nucleic acid-locked beacon of any one of claims 1-7, wherein the signaling nucleic acid sequence further comprises a barcode nucleic acid sequence and/or a primer site for amplifying said barcode nucleic acid sequence, and wherein the detectable signal is said amplified barcode nucleic acid sequence.

9. The nucleic acid-locked beacon of any one of claims 1-8, wherein the signaling nucleic acid sequence is from 4 to 100 nucleotides in length; and wherein the blocking nucleic acid is from 4 to 20 nucleotides in length.

10. The nucleic acid-locked beacon of claim 9. wherein the signaling nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 6, 8. or 10; and wherein the blocking nucleic acid sequence comprises 80% similarity or more to any one of SEQ ID NOs: 7, 9, or 14-15.

11. The nucleic acid-locked beacon of any one of claims 1-10. wherein, when the cleavable domain is intact: the nucleic acid portion of the beacon forms a secondary structure selected from a hairpin structure and a circularized structure; and the blocking nucleic acid sequence and the signaling nucleic acid sequence are at least partially hybridized.

12. The nucleic acid-locked beacon of claim 11, wherein, when the cleavable domain is intact, the blocking nucleic acid sequence and the signaling nucleic acid sequence are at least partially hybridized under a given set of conditions, and further wherein, upon cleavage of the cleavable domain by the enzyme, the blocking nucleic acid sequence and the signaling sequence at least partially denature under said given set of conditions; and wherein the given set of conditions comprise temperature, pH, and/or salt concentration.

13. The nucleic acid-locked beacon of claim 12, wherein at least partial denaturation of the blocking nucleic acid sequence and signaling nucleic acid sequence allows the signaling nucleic acid sequence to produce a detectable signal; and wherein at least partial hybridization of the blocking nucleic acid sequence and the signaling nucleic acid sequence at least partially reduces production of the detectable signal by the signaling nucleic acid sequence.

14. The nucleic acid-locked beacon of any one of claims 1-13. wherein the nucleic acid- locked beacon further comprises a linker between the cleavable domain and the blocking nucleic acid.

15. The nucleic acid-locked beacon of claim 14, wherein a portion or all of the linker comprises a bifunctional molecule, such as a PEG-based linker or a nucleic acid based linker.

16. The nucleic acid-locked beacon of any one of claims 1-15, wherein the nucleic acid- locked beacon comprise one or more non-naturally occurring amino acids or nucleic acids; and wherein the non-naturally occurring amino acids or nucleic acids make the beacon more stable and/or more resistant to degradation.

17. The nucleic acid-locked beacon of any one of claims 1-16, wherein the beacon is barcoded.

18. The nucleic acid-locked beacon of any one of claims 1-17, wherein the beacon is associated with a nanostructure such as a nanoparticle or a silica-based microparticle.

19. A composition comprising tw o or more nucleic acid-locked beacons of claim 1 , wherein the two or more nucleic acid-locked beacons comprise different peptide sequences; and wherein the two or more nucleic acid-locked beacons allows for detection of two or more different enz mes in the same assay (multiplex).

20. A method of detecting an enzyme of interest in a sample, the method comprising: a) exposing the enzyme of interest to the nucleic acid-locked beacon of claim 1, wherein said exposure occurs under conditions which allows the enzyme to recognize and cleave the cleavable domain, wherein cleavage of the cleavable domain allows the signaling sequence to produce a detectable signal; and b) detecting the signal produced by the signaling nucleic acid sequence, thereby detecting the enzyme of interest.