Mirna detection system

Abstract

The present invention provides compositions and methods for detecting miRNA in a biological sample. The compositions and methods employ a polynucleotide substrate and that is cleaved by a CRISPR / CAS system upon sensing of a miRNA in a sample, thus releasing particles attached to the polynucleotide substrate. These particles (e.g., gold nanoparticles) can then be detected, e.g., by dark field microscopy, to identify the presence of miRNA in the sample.

Description

[0001] miRNA DETECTION SYSTEM

[0002] SEQUENCE LISTING

[0003] This application contains a Sequence Listing which has been filed electronically in Extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 15, 2025, is named 51752-005WO2_Sequence_Listing_4_15_25.XML and is 22,905 bytes in size.

[0004] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support from the National Institute of Health (NIH) grants R21 CA217662 and R01 GM138778. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] MicroRNAs (miRNAs) are a class of small non-coding RNAs, typically 18-24 nucleotides in length, which exert negative regulation on gene expression. They play pivotal roles in numerous cellular processes, and aberrant miRNA expression has been implicated in various human cancers, including breast, lung, pancreatic, gastric, and colorectal cancers. In addition, several miRNAs have been reported as potential biomarkers for early cancer detection and prognosis.

[0008] However, the detection and analysis of miRNAs pose significant technical challenges. Amplification-based methods, such as reverse transcription-quantitative polymerase chain reaction (RT- qPCR), often encounter difficulties in primer design due to the short length of target miRNAs. While effective, hybridization-based techniques like Northern blots and microarrays are less feasible for clinical use due to their requirement for large sample volumes. Next-generation sequencing offers the advantage of simultaneously detecting multiple miRNAs and identifying novel ones. However, complex sample preparation, long assay times, and expensive operational costs hinder its application in clinical settings. Moreover, these conventional methods are primarily suitable for well-equipped laboratory environments rather than point-of-care testing.

[0009] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems have recently gained great attention for nucleic acid detection because of their advantages, such as rapid and direct detection, superior specificity (single-base mismatch), and isothermal reaction. Moreover, the CRISPR / CAS system showed attomolar sensitivity when the target RNAs were amplified by recombinase polymerase amplification (RPA). Although the target amplification step can increase sensitivity, this step could introduce false-negative or false-positive results due to short lengths of miRNAs and increase the assay time. For these reasons, researchers have developed target amplification-free methods to detect miRNAs using the CRISPR / CAS system. The methods used three main readout techniques: fluorescence, colometry, and electrochemistry. Among these readout techniques, fluorescence readout is one of the most widely used techniques. Recently, droplet-digital Cas13a assay using fluorescence signal demonstrated attomolar sensitivity without target amplification. On the other hand, detecting fluorescent signals usually requires bulk and expensive equipment. Detecting the signal using colorimetry is an attractive way because the signal can be measured by the naked eye. Even though the signal can be easily measured by colorimetry, this readout is difficult to quantify accurately. Electrochemical sensors are relatively simple and cost-effective but require frequent calibration due to their instability. Thus, it is essential to develop a simple, stable, quantifiable, and cost- effective readout technique for CRISPR / Cas-based miRNA biosensors.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect, the invention features a method of detecting a microRNA (miRNA). The method includes the step of (a) contacting a sample that includes the miRNA with (i) a first particle conjugated to a first polynucleotide; (ii) a second particle conjugated to a second polynucleotide; and (iii) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide. The method further includes the step of (b) contacting the sample with a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and a CRISPR associated (CAS) protein. The method further includes the steps of (c) allowing the crRNA to hybridize to the miRNA, thereby activating the CAS protein to cleave the bridge polynucleotide; and (d) detecting the first particle that is conjugated to the first polynucleotide.

[0012] In some embodiments, the first polynucleotide and second polynucleotide are a single polynucleotide integral with the bridge polynucleotide.

[0013] In some embodiments, the first polynucleotide and the second polynucleotide are separate polynucleotides, and the bridge polynucleotide includes a first portion that hybridizes to the first polynucleotide and a second portion that hybridizes to the second polynucleotide.

[0014] In some embodiments, the bridge polynucleotide includes a target cleavage sequence that is cleaved by the CAS protein. The bridge polynucleotide may be DNA, RNA, or a combination thereof. In some embodiments, the bridge polynucleotide includes RNA.

[0015] In some embodiments, the bridge polynucleotide includes a spacer region 5’ and / or 3’ of the target cleavage sequence. In some embodiments, the spacer region includes a polyT sequence. In some embodiments, the bridge polynucleotide includes a polyT spacer 5’ of the target cleavage sequence. In some embodiments, the bridge polynucleotide includes a polyT spacer 3’ of the target cleavage sequence.

[0016] In some embodiments, the method further includes, following step (c), separating the first particle from the sample. For example, separating may include immobilizing the first particle, e.g., with a capture agent that binds to the first polynucleotide.

[0017] In some embodiments, the capture agent is conjugated to a glass slide. For example, the capture agent may be biotinylated, and the glass slide may be coated with streptavidin.

[0018] In some embodiments, the capture agent is a third polynucleotide that hybridizes to the first polynucleotide.

[0019] In some embodiments, the method includes incubating the sample with the capture agent for 1 to 30 minutes (e.g., for 10 to 15 minutes).

[0020] In some embodiments, the first particle includes a plurality of the first polynucleotides conjugated to the first particle.

[0021] In some embodiments, the first particle is a metallic nanoparticle, such as a gold nanoparticle. In some embodiments, the gold nanoparticle is from 10 nm to 200 nm (e.g., 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, e.g., 60 nm) in diameter.

[0022] In some embodiments, the first polynucleotide includes a thiol. In some embodiments, the first particle is treated with tris(2-carboxyethyl)phosphine (TCEP) and conjugated to the thiol of the first polynucleotide.

[0023] In some embodiments, the method further includes, following step (c), separating the second particle from the sample. In some embodiments, separating includes immobilizing the second particle.

[0024] In some embodiments, the second particle is a magnetic bead.

[0025] In some embodiments, the magnetic bead is coated with streptavidin.

[0026] In some embodiments, the second polynucleotide is biotinylated.

[0027] In some embodiments, the separating includes using a magnet to immobilize the magnetic bead.

[0028] In some embodiments, the magnetic bead is from 100 nm to 500 nm (e.g., 100 nm, 200 nm, 300 nm, 400 nm, or 500 nm, e.g., 200 nm) in diameter.

[0029] In some embodiments, the method includes separating the second particle from the sample prior to separating the first particle from the sample.

[0030] In some embodiments, the method includes separating the second particle from the sample and saving a supernatant that includes the first particle.

[0031] In some embodiments, the second particle includes a plurality of the second polynucleotides conjugated to the second particle.

[0032] In some embodiments, the sample includes a plurality of the first particles and a plurality of the second particles.

[0033] In some embodiments, step (d) includes detecting the first particle with dark-field imaging.

[0034] In some embodiments, the step (d) includes side illumination of a light-emitting diode (LED) light.

[0035] In some embodiments, step (d) includes side illumination light coupled with total internal reflection to produce scattered light.

[0036] In some embodiments, step (d) includes detecting the first particle for 1 to 5 minutes (e.g., 1 , 2, 3, 4, or 5 minutes).

[0037] In some embodiments, the CAS protein is CAS13a.

[0038] In some embodiments, the miRNA is miR-21 -5p, miR-9-5p, or miR-421 .

[0039] In some embodiments, the first polynucleotide, the second polynucleotide, and / or the bridge polynucleotide is a deoxyribonucleotide (DNA), a ribonucleotide (RNA), or a combination thereof.

[0040] In some embodiments, the first polynucleotide is DNA, the second polynucleotide is DNA, and the bridge polynucleotide is RNA.

[0041] In some embodiments, the method further includes, prior to step (c), forming a complex with the crRNA and the CAS protein.

[0042] In some embodiments, a level of expression of the miRNA in the sample is associated with cancer, and the method includes diagnosing a subject with cancer upon detection of the level of expression of the miRNA above a predetermined threshold in the sample. For example, in some embodiments, the level is increased by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, e.g., relative to a baseline, e.g., a control sample, e.g., sample of a subject without cancer or of the subject prior to having cancer.

[0043] In some embodiments, the cancer is breast cancer or ovarian cancer.

[0044] In some embodiments, the method further includes treating the cancer.

[0045] In another aspect, the invention features a system or kit for detecting miRNA . The system or kit includes one or more of (a) a first particle conjugated to a first polynucleotide; (b) a second particle conjugated to a second polynucleotide; (c) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide; (d) a crRNA; (e) a CAS protein, wherein the miRNA is configured to hybridize to the crRNA to activate the CAS protein to cleave the bridge polynucleotide; and (f) a dark field microscope.

[0046] In another aspect, the invention features a composition that includes (i) a first particle conjugated to a first polynucleotide; (ii) a second particle conjugated to a second polynucleotide; and (iii) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide.

[0047] In some embodiments, the first polynucleotide and second polynucleotide are a single polynucleotide integral with the bridge polynucleotide.

[0048] In some embodiments, the first polynucleotide and the second polynucleotide are separate polynucleotides, and the bridge polynucleotide includes a first portion that hybridizes to the first polynucleotide and a second portion that hybridizes to the second polynucleotide.

[0049] In some embodiments, the composition further includes a miRNA.

[0050] In some embodiments, the composition further includes a crRNA and a CAS protein, wherein the CAS protein is configured to cleave the bridge polynucleotide.

[0051] In some embodiments, the first particle is a magnetic bead.

[0052] In some embodiments, the second particle is a gold nanoparticle.

[0053] In another aspect, the invention features a composition that includes (i) a metal particle conjugated to a first polynucleotide; and (ii) a substrate conjugated to a capture agent, wherein the capture agent is attached to the first polynucleotide.

[0054] In some embodiments, the capture agent is a second polynucleotide that is hybridized to the first polynucleotide.

[0055] In some embodiments, the metal particle includes a plurality of the first polynucleotides.

[0056] In some embodiments, at least one of the plurality of first polynucleotides is hybridized to a portion of a bridge polynucleotide cleaved by a CAS protein.

[0057] In some embodiments, the metal particle is a gold nanoparticle.

[0058] In some embodiments, the substrate is a glass slide.

[0059] In some embodiments, the capture agent is biotinylated, and the glass slide is coated with streptavidin.

[0060] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0062] FIGS. 1A-1G are a schematic drawing, gel, micrograph, and photographs showing CRISPR / Cas13a-assisted magnetic nanoparticle (MGNP)-dark-field (DF) assay. (FIG. 1A) Schematic diagram for the CRISPR / Cas13a-assisted MGNPDF assay. In the presence of target miR-21 -5p, the hybridization of the crRNA to miR-21 -5p activates Cas13a proteins. Activated Cas13a proteins then cleave the bridge linkers between magnetic beads and gold nanoparticles (AuNPs). Subsequent magnetic separation of the magnetic beads releases AuNPs. These released AuNPs are captured onto a glass slide with black PDMS wells. The number of released AuNPs is quantified using a custom-built, portable DF imaging system. The entire assay can be completed within 30 min. (FIG. 1B) Bacterial expression and purification of LwaCas13a. Purified LwaCas13a was shown by SDS-PAGE, followed by silver staining. (FIG. 1 C) Synthesis of miR-21 -5p crRNA by in vitro transcription was confirmed by denaturing PAGE and SYBR gold staining. (FIG. 1D) Scanning electron micrograph (SEM) of MGNP. Scale bar, 100 nm. (FIG. 1 E). Photograph of the portable DF imaging device. The overall size is 140 mm (L) x 85 mm (W) x 250 mm (H). (FIG. 1 F) 10x objective lens and slide glass holders coupled with a white LED. (FIG. 1G) Representative zoomed-in DF images of AuNPs detached from magnetic beads by mock (left) or miR-21 -5p treatment (right). Scale bar,100 pm.

[0063] FIGS. 2A and 2B are graphs showing Dynamic Light Scattering (DLS) measurement results for mixture of magnetic beads (MBs) and gold nanoparticles (AuNPs) without (FIG. 2A) or with (FIG. 2B) a linker.

[0064] FIGS. 3A-3E are DF images and quantification showing a CRISPR / Cas13a-assisted MGNP-DF assay. (FIG. 3A) DF images of released AuNPs with transparent and black PDMS (left) chambers. Comparison of plot profiles between PDMS and black PDMS chambers (right). Scale bar, 100 pm. (FIG. 3B) Comparison of the size of magnetic beads. Magnetic beads with a 200 nm diameter showed a higher signal of AuNPs than magnetic beads with a 1 pm diameter. *P < 0.05 compared with 200 nm and 1 pm, as assessed by unpaired t-test. Bar graphs are shown as mean ± SD. (FIG. 3C) Comparison of the size of AuNPs. AuNPs with a 60 nm diameter showed a higher signal than AuNPs with an 80 nm diameter by miR-21 -5p-mediated Cas13a activation, ns, not significant; ****P < 0.0001 compared with the mock sample, as assessed by two-way ANOVA with Bonferroni's multiple comparisons tests. (FIG. 3D) The number of AuNPs released from 3 different designs of MGNPs (1 ; linker only, 2; bridge without poly-T, 3; bridge with poly-T) depending on the amount of miR-21 -5p. The reaction volume was 20 pl. Error bars are shown as mean ± SD. (FIG. 3E) The number of AuNPs detached from magnetic beads depending onthe Cas13a reaction time. Error bars are shown as mean ± SD.

[0065] FIG. 4 is a set of dark-field (DF) images of gold nanoparticles (AuNPs) of different sizes (50 nm, 60 nm, and 80 nm) captured using a portable DF imaging system. Scare bar, 100 pm.

[0066] FIGS. 5A-5C are schematic drawings showing hybridization strategies for linking magnetic beads (MBs) and gold nanoparticles (AuNPs) using nucleic acid linkers. (FIG. 5A) Direct linking of MBs and AuNPs with a single-stranded DNA / RNA / DNA linker. B-C. Sandwich hybridization with bridges without (FIG. 5B) or with (FIG. 5C) poly-T. DNA and RNA sequences are denoted in black and red, respectively. Poly-T sequences are highlighted in blue.

[0067] FIGS. 6A-6C are a set of dark field images and graphs showing an evaluation of sensitivity and specificity of the CRISPR / Cas13a-assisted MGNP-DF assay. (FIGS. 6A and 6B) Measurement of released AuNPs by seven different concentrations of miR-21 -5p. (FIG. 6A) Representative zoomed-in images for the 4 different target miRNA amounts. Scale bar, 100 pm. (FIG. 6B) Quantitative analysis for the numbers of released AuNPs. The dotted line indicates the number of mean + 3 x standard deviation (s.d.) of the mock sample. Error bars are shown as mean ± s.d. from three independent experiments. (FIG. 6C) Detection specificity was evaluated using wildtype (WT), single-mismatch (SM), and doublemismatches (DM) of miR-21 -5p, miR-421 , and miR-9-5p. Bar graphs are shown as mean ± SD from the three experiments. ****P < 0.0001 compared miR-21 -5p (WT) sample, as assessed by two-way ANOVA with Bonferroni's multiple comparisons test.

[0068] FIGS. 7A and 7B are a table and graph showing a calculation of the limit of the detection. (FIG. 7A) Signal values for different concentrations of miR-21 -5p and negative control (mock) to determine the limit of detection (LOD) threshold by the mean + 3 times the standard deviation. Based on the threshold value (510.9), we determined the LOD as 0.5 fmole (25 pM). (FIG. 7B) Bar graph showing the signal values from different miR-21 -5p concentrations and the LOD threshold value.

[0069] FIGS. 8A-8D are a set of DF images and graphs showing miR-21 -5p detection from breast cancer cell lines. (FIGS. 8A) Representative zoomed-in DF images of released AuNPs by miR-21 -5p- activated Cas13a. Scale bar, 100 pm. (FIGS. 8B) The number of released AuNPs in five different breast cancer cell lines. Bar graphs are shown as mean ± SD from the three independent experiments. (FIGS. 8C) Relative abundances of miR-21 -5p, as analyzed by RT-qPCR. U6 snRNA levels were used as an internal control. Bar graphs are shown as mean ± SD from the three independent experiments. (FIGS. 8D) Dot plot for correlation coefficient. The number of released AuNPs from the CRISPR / Cas13a-assisted MGNP-DF assay and the relative abundance from RT-qPCR are indicated in the y- and x-axis, respectively. The black dashed line indicates the best linear fit. The two blue dashed lines indicate the 95% confidence interval.

[0070] DETAILED DESCRIPTION

[0071] MicroRNAs (miRNAs) are short (about 18-24 nucleotides) non-coding RNAs and have emerged as potential biomarkers for various diseases, such as cancer. miRNAs are challenging to detect due to their short length and, in some instances, low abundance. The present invention includes a new miRNA detection system to detect miRNA in a sample. The system employs a cleavable polynucleotide substrate that is tethered to particles that can be immobilized. Upon detection of the miRNA in a sample, a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and a CRISPR associated (CAS) protein complex can be activated by the presence of the miRNA in the sample, thereby cleaving the polynucleotide substrate. Upon cleavage, one or more of the particles tethered to the polynucleotide substrate can be detected, thereby confirming the presence of the miRNA in the sample (see, e.g., FIG. 1 A for a schematic diagram of the method).

[0072] The invention is advantageous for several reasons. It is essential to develop a rapid, selective, and accurate miRNA detection assay using a simple, affordable system. Herein is provided a CRISPR / Cas-based miRNA biosensor that may be combined with point-of-care dark-field (DF) imaging. The combination of CRISPR / Cas, particle-based detection (e.g., gold nanoparticles), and dark field imaging demonstrates amplification-free detection of miRNA in less than 30 minutes at a limit of detection of 500 attomole and with single-base specificity. The CRISPR / CAS-assisted biosensor can achieve rapid, selective, and accurate detection of miRNAs with simple equipment, thus providing a potential application for cancer diagnosis. These compositions, systems, and methods have been shown to demonstrate rapid, selective, and accurate detection of miRNAs using simple equipment, suggesting the potential for early point of care tumor diagnosis.

[0073] Methods, Compositions, and Systems for miRNA Detection

[0074] The methods and systems described herein are useful for detecting a microRNA (miRNA), e.g., in a biological sample. The method described herein includes a step of (a) contacting a sample that includes the miRNA with (i) a first particle conjugated to a first polynucleotide; (ii) a second particle conjugated to a second polynucleotide; and (iii) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide. The method further includes the step of (b) contacting the sample with a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and a CRISPR associated (CAS) protein. The method further includes the steps of (c) allowing the crRNA to hybridize to the miRNA, thereby activating the CAS protein to cleave the bridge polynucleotide; and (d) detecting the first particle that is conjugated to the first polynucleotide. The first particle may be, for example, a metallic particle (e.g., a gold particle, e.g., a gold nanoparticle). Metallic nanoparticles may be easily detected through a variety of common techniques, such as colorimetric methods, optical microscopy, dark field imaging, total internal reflection fluorescence (TIRF) microscopy, and surface-enhanced Raman spectroscopy (SERS).

[0075] The bridge polynucleotide may be a single polynucleotide (e.g., a single stranded polynucleotide, e.g., a single stranded RNA). For example, in some embodiments, the first polynucleotide and second polynucleotide are a single polynucleotide integral with the bridge polynucleotide. The sample may include a plurality of bridge polynucleotides, a plurality of the first particles, and a plurality of the second particles.

[0076] Alternatively, the bridge polynucleotide may include two or more polynucleotides, e.g., as a double stranded polynucleotide or with the bridge polynucleotide hybridized to the first and second polynucleotides that are each conjugated to a particle. For example, in some embodiments, the first polynucleotide and the second polynucleotide are separate polynucleotides, and the bridge polynucleotide includes a first portion that hybridizes to the first polynucleotide and a second portion that hybridizes to the second polynucleotide (see, e.g., FIGS. 5A-5C for different bridge polynucleotide arrangements). In some embodiments, the first polynucleotide, the second polynucleotide, and / or the bridge polynucleotide is a deoxyribonucleotide (DNA), a ribonucleotide (RNA), or a combination thereof. In some embodiments, the first polynucleotide is DNA, the second polynucleotide is DNA, and the bridge polynucleotide is RNA.

[0077] The bridge polynucleotide may include a target cleavage sequence that is cleaved by the CAS protein. Thus, upon detection of the miRNA in the sample, a crRNA / CAS complex can cleave the target sequence of the bridge polynucleotide, thus breaking the bridge, and releasing the connection between the tethered particles. The bridge polynucleotide may be DNA, RNA, or a combination thereof. In some embodiments, the bridge polynucleotide includes RNA. The bridge polynucleotide may include a spacer region 5’ and / or 3’ of the target cleavage sequence. In some embodiments, the spacer region includes a polyT sequence. In some embodiments, the bridge polynucleotide includes a polyT spacer 5’ of the target cleavage sequence. In some embodiments, the bridge polynucleotide includes a polyT spacer 3’ of the target cleavage sequence.

[0078] In some embodiments, the method further includes forming a complex with the crRNA and the CAS protein, e.g., prior to contacting the sample with the CRISPR / CAS complex.

[0079] Following cleavage of the bridge polynucleotide, the method may further include separating the first particle (e.g., the gold nanoparticle) from the sample. For example, separating may include immobilizing the first particle, e.g., with a capture agent that binds to the first polynucleotide that is conjugated to the first particle.

[0080] The capture agent may be any suitable probe that binds to the polynucleotide or portion thereof that is conjugated to the first particle. For example, in some embodiments, the capture agent is a polynucleotide that hybridizes to the polynucleotide conjugated to the particle. In some embodiments, the capture agent is a protein that binds to the polynucleotide. The capture agent may be immobilized to a surface, e.g., a glass surface, such that that the particle (e.g., gold nanoparticle) is immobilized at or near the surface to which the capture agent is attached, thereby providing improved and spatially resolved detection.

[0081] The capture agent may be conjugated to a glass slide, e.g., by using a biotinylated capture agent that is attached to a streptavidin coated glass slide. The capture agent may be incubated with the sample containing the miRNA (e.g., with the cleaved bridge polynucleotide) for any suitable amount of time such that the polynucleotide conjugated to the particle is sufficiently immobilized via its attachment with the capture agent. For example, the capture agent may be incubated with the sample for at least 1 minute (e.g., at least 10 minutes, 20 minutes, 30 minutes, or more, e.g., 1 to 30 minutes, e.g., 10 to 15 minutes). Using a glass slide to immobilize the particle is advantageous in that it can then subsequently be used for imaging without transferring the sample to a different surface or substrate.

[0082] The first particle (e.g., gold nanoparticle) may include a plurality of polynucleotides (e.g., the same or different polynucleotides) conjugated to the particle. By including a plurality of polynucleotides attached to the particle, this may increase the chance for the polynucleotide to interact with the capture agent.

[0083] In some embodiments, the method further includes separating the second particle from the sample. Separating the second particle allows a user to wash to enrich he sample in the first particles by removing uncleaved bridging polynucleotides and portions of the cleaved bridge polynucleotide that lack the first (e.g., metallic, e.g., gold) particles. In some embodiments, separating includes immobilizing the second particle. The second particle may be a magnetic particle, and the separating includes immobilizing the magnetic particles by applying a magnetic force. Separation of the second particle may be performed, for example, separating the first particles and / or detection of the first particles. In some embodiments, the method includes separating the second particle from the sample and saving a supernatant that includes the first particle, e.g., for subsequent use (e.g., detection and / or analysis).

[0084] Following cleavage of the bridge polynucleotide (prior to and / or after enrichment) the method includes detecting the first particles (e.g., gold nanoparticles). Metallic nanoparticles may be easily detected through a variety of common techniques, such as colorimetric methods, optical microscopy, dark field imaging, TIRF microscopy, and SERS. In some embodiments, the detection includes detecting the first particle with dark-field imaging. In some embodiments, the detection includes side illumination of an LED light. In some embodiments, the detection includes side illumination light coupled with total internal reflection to produce scattered light. The particles may be detected for any suitable amount of time, e.g., an amount of time sufficient to generate a signal to identify the presence or absence of the particles. The particles may be detected for at least 1 minute, e.g., at least 5 minutes, e.g., at least 10 minutes, e.g., at least 20 minutes, or more. In some embodiments, the particles are detected for 1 to 5 minutes (e.g., 1 , 2, 3, 4, or 5 minutes).

[0085] Any suitable miRNA can be used herein with the systems and methods described herein. In some embodiments, the miRNA is miR-21 -5p, miR-9-5p, or miR-421. In some embodiments, the miRNA is a miRNA that is present or upregulated in a cancer cell. For example, a miRNA that is upregulated in a cancer cell may be an miRNA that exhibits an increased level of expression (e.g., by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, e.g., relative to a baseline, e.g., a control sample, e.g., sample of a subject without cancer or of the subject prior to having cancer). IN some embodiments, the miRNA is a miRNA that is present in a cancerous cell but not in a non-cancerous or control cell.

[0086] In some embodiments, a level of expression of the miRNA in the sample is associated with cancer, and the methods described herein include diagnosing a subject with cancer, e.g., upon detection of the level of expression of the miRNA above a predetermined threshold in the sample. For example, in some embodiments, the level is increased by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, e.g., relative to a baseline, e.g., a control sample, e.g., sample of a subject without cancer or of the subject prior to having cancer. In some embodiments, the cancer is breast cancer or ovarian cancer. In some embodiments, the method further includes treating the cancer. In some embodiments, the methods described herein include treating a subject for cancer where the subject or a sample thereof has previously been identified as being cancerous due to detection of the presence or increased expression of a miRNA characteristic of a cancer.

[0087] Also featured herein is a system or kit for detecting miRNA. The system or kit includes one or more of (a) a first particle (e.g., a magnetic bead) conjugated to a first polynucleotide; (b) a second particle (e.g., a gold nanoparticle) conjugated to a second polynucleotide; (c) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide; (d) a crRNA; (e) a CAS protein, wherein the miRNA is configured to hybridize to the crRNA to activate the CAS protein to cleave the bridge polynucleotide; and (f) a dark field microscope. Any suitable microscope may be used in a system or kit as described herein. In some embodiments, the microscope is a hand-held microscope. In some embodiments, the microscope employs one or more of colorimetric methods, optical microscopy, dark field imaging, total internal reflection, TIRF microscopy, and SERS. In some embodiments, the microscope employs dark-field imaging, e.g., by side illumination of an LED, e.g., coupled with total internal reflection to produce scattered light.

[0088] Also featured herein is a composition that includes (i) a first particle (e.g., a magnetic bead) conjugated to a first polynucleotide; (ii) a second particle (e.g., a metallic particle, e.g., a gold nanoparticle) conjugated to a second polynucleotide; and (iii) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide. In some embodiments, the first polynucleotide and second polynucleotide are a single polynucleotide integral with the bridge polynucleotide. In some embodiments, the first polynucleotide and the second polynucleotide are separate polynucleotides, and the bridge polynucleotide includes a first portion that hybridizes to the first polynucleotide and a second portion that hybridizes to the second polynucleotide. In some embodiments, the composition further includes a miRNA. In some embodiments, the composition further includes a crRNA and a CAS protein, wherein the CAS protein is configured to cleave the bridge polynucleotide.

[0089] Also featured herein is a composition that includes (i) a metal particle (e.g., a gold nanoparticle) conjugated to a first polynucleotide; and (ii) a substrate (e.g., a glass slide) conjugated to a capture probe, wherein the capture probe is attached to the first polynucleotide. In some embodiments, the capture probe is a second polynucleotide that is hybridized to the first polynucleotide. In some embodiments, the metal particle includes a plurality of the first polynucleotides. In some embodiments, at least one of the plurality of first polynucleotides is hybridized to a portion of a bridge polynucleotide cleaved by a CAS protein. In some embodiments, the capture probe is biotinylated, and the glass slide is coated with streptavidin.

[0090] CRISPR / CAS Systems

[0091] The compositions, systems, and methods described herein employ a CRISPR / CAS system to cleave a bridging polynucleotide, e.g., upon detection of a miRNA. crRNA is a derived from an RNA transcript of the CRISPR locus. The crRNA is further processed by a CAS protein, a protein with helicase and nuclease activity. Upon formation of a complex between the crRNA and the CAS protein, the crRNA guides the complex to the target polynucleotide sequence (e.g., the bridging polynucleotide) for cleavage. In some embodiments, the CRISPR / CAS system further includes a guide RNA (gRNA), e.g., depending on the type of CRISPR / CAS system to guide the complex to its target.

[0092] In some embodiments, the CAS protein is CAS13a. Cas13a is an RNA-guided, RNA-activated nuclease, meaning it cleaves RNA molecules based on a specific RNA sequence. It's a type Vl-A CRISPR enzyme, which is typically used for RNA detection, editing, and knockdown. Cas13a recognizes single-stranded RNA targets and activates its ribonuclease activity, which is crucial for its function in RNA-based applications.

[0093] In a typical CRISPR / CAS system, an endonuclease is directed to a target nucleotide sequence (e.g., a bridging polynucleotide) by sequence-specific, non-coding guide RNAs that target single- or double-stranded DNA sequences. Three classes (l-lll) of CRISPR systems have been identified. The class II CRISPR systems use a single CAS endonuclease (rather than multiple CAS proteins). One class II CRISPR system includes a type II CAS endonuclease such as Cas9, a CRISPR RNA (crRNA), and a trans-activating crRNA (tracrRNA). The crRNA contains a guide RNA, which is typically an about 20- nucleotide RNA sequence that corresponds to a target DNA sequence. The crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure which is cleaved by RNase III, resulting in a crRNA / tracrRNA hybrid. The RNAs serve as guides to direct CAS proteins to silence specific DNA / RNA sequences, depending on the spacer sequence. See, e.g., Horvath et al., Science 327:167-170, 2010; Makarova et al., Biology Direct 1 :7, 2006; Pennisi, Science 341 :833-836, 2013, which are herein incorporated by reference. The target DNA sequence must generally be adjacent to a protospacer adjacent motif (PAM) that is specific for a given CAS endonuclease; however, PAM sequences appear throughout a given genome. Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5’ from) the PAM site. Other CAS nucleases include, for example, a Cas12b nuclease or a CasX nuclease.

[0094] Another class II CRISPR system includes the type V endonuclease Cpf 1 , which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpf1 -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words a Cpf 1 system requires only the Cpf 1 nuclease and a crRNA to cleave the target DNA sequence. Cpf 1 endonucleases, are associated with T-rich PAM sites, e.g., 5’-TTN. Cpf 1 can also recognize a 5’-CTA PAM motif. Cpf 1 cleaves the target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5’ overhang, for example, cleaving a target DNA with a 5- nucleotide offset or staggered cut located 18 nucleotides downstream from (3’ from) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5- nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA.

[0095] CRISPR arrays can be designed to contain one or multiple guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al., Science 339:819-823, 2013; Ran et al., Nature Protocols 8:2281 -2308, 2013. At least about 16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNA cleavage to occur. In practice, guide RNA sequences are generally designed to have a length of between 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and complementarity to the targeted gene or nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. CRISPR / CAS based cleavage has also been achieved using a chimeric single guide RNA (sgRNA), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective in target clevage; see, for example, Hendel et al., Nature Biotechnol. 985-991 , 2015.

[0096] Particles

[0097] The polynucleotides and / or capture agents described herein may be conjugated to one or more particles, e.g., a magnetic particle, a metallic particle, or a bead. In some embodiments, the polynucleotide and / or capture agent is conjugated to a plurality of particles. In some embodiments, a particle is conjugated to a plurality of capture agents and / or polynucleotides.

[0098] Metallic particles include at least one metallic component with the particle. The magnetic particle may be a gold or silver particle, e.g., a gold nanoparticle. Gold nanoparticles are advantageous for dark field imaging in order to provide quick and facile detection. Further, metal nanoparticles are useful as they may be detected at the single particle level.

[0099] Magnetic particles include at least one component that is responsive to a magnetic force. A magnetic particle may be entirely magnetic or may contain components that are non-magnetic. A magnetic particle may be a magnetic bead, e.g., a substantially spherical magnetic bead. The magnetic particle may be entirely magnetic or may contain one or more magnetic cores surrounded by one or more additional materials, such as, for example, one or more functional groups and / or modifications for binding one or more target molecules. In some examples, a magnetic particle may contain a magnetic component and a surface modified with one or more silanol groups.

[0100] A particle, e.g., a magnetic particle or a bead, may be porous, non-porous, hollow, solid, semisolid, semi-fluidic, fluidic, and / or a combination thereof. In some instances, a particle, e.g., a bead, may be dissolvable or degradable. In some cases, a particle, e.g., a bead, may not be degradable. In some embodiments, the bead is composed of crosslinked agarose, e.g., SEPHAROSE®.

[0101] A particle, e.g., a magnetic particle, metallic particle, or a bead, may include natural and / or synthetic materials. For example, a particle, e.g., a bead, can include a natural polymer, a synthetic polymer or both natural and synthetic polymers. Examples of natural polymers include proteins and sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum, corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate, or natural polymers thereof. Examples of synthetic polymers include acrylics, nylons, silicones, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), polyethylene oxide), polyethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and / or combinations (e.g., copolymers) thereof. Beads may also be formed from materials other than polymers, including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and others.

[0102] Cross-linking may be permanent or reversible, depending upon the particular cross-linker used. Reversible cross-linking may allow for the polymer to linearize or dissociate under appropriate conditions. In some cases, reversible cross-linking may also allow for reversible attachment of a material bound to the surface of a bead.

[0103] Particles, e.g., beads or magnetic particles, may be of uniform size or heterogeneous size. In some cases, the diameter of a particle, e.g., a bead, may be at least about 1 pm, 5 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 250 pm, 500 pm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or greater. In some cases, a particle, e.g., a bead, may have a diameter of less than about 1 pm, 5 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 250 pm, 500 pm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or less. In some cases, a particle, e.g., a bead, may have a diameter in the range of about 40-75 pm, 30-75 pm, 20-75 pm, 40-85 pm, 40-95 pm, 20-100 pm, 10-100 pm, 1 -100 pm, 20-250 pm, or 20-500 pm, 500 pm-1 mm, 1 mm-2 mm, 1 -5 mm, or 1 -10 mm.

[0104] In some embodiments, the first particle is a metallic nanoparticle, such as a gold nanoparticle. In some embodiments, the gold nanoparticle is from 10 nm to 200 nm (e.g., 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, e.g., 60 nm) in diameter. In some embodiments, the second particle is a magnetic bead. In some embodiments, the magnetic bead is from 100 nm to 500 nm (e.g., 100 nm, 200 nm, 300 nm, 400 nm, or 500 nm, e.g., 200 nm) in diameter.

[0105] Particles may be of any suitable shape. Examples of particles, e.g., magnetic particles or beads, shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.

[0106] Conjugation and Attachment Groups

[0107] In some embodiments, one or more chemical moieties are used to attach two or more components. An attachment may be covalent or noncovalent attachment. For example, a non-covalent attachment may include an antibody / antigen interaction. A covalent attachment may include a linker that directly conjugates two components.

[0108] In some embodiments, a linker is used to conjugate two or more components used in a composition or method described herein. For example, a linker may be used to conjugate a polynucleotide to a particle, a capture agent to a surface, or any combination or variation thereof. In some embodiments, the polynucleotide is conjugated to the first capture agent with a chemical linker. The chemical linker may be conjugated to a 3’ end or a 5’ end of the polynucleotide. Alternatively, the chemical linker may be conjugated to an interior region of the polynucleotide.

[0109] In some embodiments, the polynucleotide is conjugated to a particle. The particle may be, for example, a magnetic particle or a bead. The bead may be, e.g., a crosslinked agarose, e.g., a SEPHAROSE®, bead. In some embodiments, a polynucleotide is conjugated directly to a particle (e.g., a metallic particle or a bead, e.g., a magnetic bead).

[0110] A chemical linker provides space, rigidity, and / or flexibility between, for example, a polynucleotide and particle and / or a surface and a capture agent. In some embodiments, a linker may be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a C-0 bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation. In some embodiments, a linker includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1- 130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1 -240, or 1 -250 atom(s);250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). In some embodiments, a linker includes no more than 250 non-hydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1- 12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1- 90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1- 230, 1 -240, or 1 -250 non-hydrogen atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-hydrogen atom(s)). In some embodiments, the backbone of a linker includes no more than 250 atoms (e.g., 1 -2, 1 -4, 1 -6, 1 -8, 1-10, 1 -12, 1-14, 1 -16, 1-18, 1 -20, 1 -25, 1 -30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1- 140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1 -240, or 1 -250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). The “backbone” of a linker refers to the atoms in the linker that together form the shortest path from one part of the conjugate to another part of the conjugate. The atoms in the backbone of the linker are directly involved in linking one part of the conjugate to another part of the conjugate. For example, hydrogen atoms attached to carbons in the backbone of the linker are not considered as directly involved in linking one part of the conjugate to another part of the conjugate.

[0111] In some embodiments, a linker may include a synthetic group derived from, e.g., a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). The chemical linker may include, e.g., triethylene glycol (TEG). In some embodiments, a linker may include one or more amino acid residues. In some embodiments, a linker may be an amino acid sequence (e.g., a 1 -25 amino acid, 1 -10 amino acid, 1 -9 amino acid, 1 -8 amino acid, 1 -7 amino acid, 1 -6 amino acid, 1 -5 amino acid, 1 -4 amino acid, 1 -3 amino acid, 1 -2 amino acid, or 1 amino acid sequence). In some embodiments, a linker may include one or more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene (e.g., a PEG unit), optionally substituted C2-C20 alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C2-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene (e.g., Ce arylene), optionally substituted C3-C15 heteroarylene (e.g., imidazole, pyridine), O, S, NRi (Ri is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C2-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C3-C15 heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.

[0112] Covalent conjugation of two or more components in a conjugate using a linker may be accomplished using well-known organic chemical synthesis techniques and methods. Complementary functional groups on two components may react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine. Site-specific conjugation to a polypeptide may accomplished using techniques known in the art.

[0113] In some embodiments, the first polynucleotide includes a thiol for conjugation to the first particle (e.g., gold nanoparticle). In some embodiments, the particle is treated with tris(2-carboxyethyl)phosphine (TCEP) and conjugated to the thiol of the polynucleotide.

[0114] In some embodiments, an antibody / antigen interaction is used to attach two or more components as described herein. For example, in some embodiments, the magnetic bead is coated with streptavidin. In some embodiments, the polynucleotide is biotinylated, e.g., to attach the magnetic bead to the polynucleotide. In some embodiments, the capture agent is conjugated to a surface, e.g., a glass slide, e.g., by using a biotinylated capture agent that is attached to a streptavidin coated glass slide.

[0115] EXAMPLES

[0116] Example 1. miRNA Detection System

[0117] Below we describe a CRISPR / Cas13a-assisted miRNA detection system via magnetic-gold nanoparticles (MGNPs) and dark-field (DF) imaging. We started with MGNP hybrids, which comprised 200 nm-sized magnetic beads and 60 nm-sized gold nanoparticles (AuNPs) conjugated by DNA / RNA linkers. In the presence of target RNAs, guide RNAs recognize and hybridize to the target. The hybridization between target miRNA and guild RNA activates CRISPR / Cas13a proteins, which cleave the bridge linkers, dissociating AuNPs from magnetic beads. We developed a portable DF imaging system to accurately quantify the released AuNPs. The total assay is finished within 30 min by a simple and short reaction time of CRISPR / Cas13a (10 min), AuNPs conjugation on a glass slide (15 min), and DF imaging (3 min). This integrated approach of CRISPR / Cas13a, MGNPs, and DF imaging enables direct detection of miR-21 -5p with single-base specificity and limits of detection (LOD) in the 500 attomole. Cross- validation of assay accuracy was performed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with small RNAs from five different breast cancer cell lines. The CRISPR / Cas1 Sa- assisted MGNP-DF assay demonstrates rapid, selective, and accurate detection of miRNAs using simple equipment, suggesting its potential for early tumor diagnosis.

[0118] RESULTS AND DISCUSSION

[0119] SYSTEM OVERVIEW OF THE CRISPR / CAS13A-ASSISTED MGNP-DF ASSA Y

[0120] In the assay design, we focused on simplicity, rapid analysis, accurate quantification, and point- of-care operation. We employed the CRISPR-Cas13a system for simple, rapid, and direct detection of target miRNA. The amplification-free detection method enables specific recognition of single target miRNAs. We used AuNPs for detection probes. AuNPs exhibit strong scattering signals, providing the ability to quantify AuNPs by DF imaging. We formed magnetic-AuNP complexes connected with a DNA / RNA / DNA bridge that can be readily cleaved by activated Cas13a. FIG. 1A shows the process of miR-21 -5p detection using the CRISPR / Cas13a-based system. In the presence of target miR-21 -5p, the hybridization of the crRNA and miR-21 -5p activates Cas13a proteins. The activated Cas13a protein then cleaves the bridge of MGNPs. Once the activated Cas13a proteins cleave the bridge, AuNPs are released and captured on a glass slide, while uncleaved complexes are easily removed by magnetic washing. The number of released AuNPs was counted by a custom-designed portable DF imaging system. The total assay is done in 30 min, including CRISPR / Cas13a reaction (10 min), AuNPs conjugation on a slide glass (15 min), and DF imaging (3 min).

[0121] We first prepared LwaCas13a and crRNA as described in our previous publication.28To induce the expression of LwaCas13a, we utilized bacteria and subsequently purified the protein using a two-step process. The first step involved nickel-nitrilotriacetic acid (Ni-NTA) purification, followed by SUMO cleavage and a second round of Ni-NTA purification. We confirmed the successful purification of LwaCas13a by performing SDS-PAGE analysis and silver staining, which revealed a distinct band corresponding to the expected size of approximately 150 kDa, indicating the protein's high integrity (FIG. 1B). To synthesize crRNA for miR-21 -5p, we employed in vitro transcription and confirmed the crRNA's integrity using denaturing PAGE and SYBR gold gel staining (FIG.1C).

[0122] Secondly, we successfully synthesized and characterized the MGNPs. MGNPs were prepared by DNA hybridization between bridge and DNA linkers immobilized on the surface of the 60 nm AuNPs and 200 nm magnetic beads. The conjugation of MGNPs was validated by the dynamic light scattering (DLS) measurement (FIG. 2A and 2B). The mixture of AuNPs and magnetic beads without bridge linkers shows two distinct peaks in the DLS measurement (FIG. 2A). The DLS result of MGNPs shows one peak as the magnetic beads and AuNPs are connected by bridge linkers (FIG. 2B). In addition, MGNPs were further validated with scanning electron microscopy (SEM), which verifies the formation of the hybrid particles more clearly (FIG. 1D).

[0123] Next, we designed the portable DF imaging system to detect and quantify the released AuNPs (Figs. 1E and 1F). The DF imaging system has overall dimensions of 140 mm in length, 85 mm in width, and 250 mm in height, making it small enough to be classified as a portable system. The compact design of the DF imaging system could be achieved by side illumination of a light-emitting diode (LED) light. Unlike the conventional DF imaging system, the side illumination does not need to use a higher numerical aperture (NA) condenser, which makes the imaging system cost-effective, compact, and reliable for a portable system. The side-illuminated light coupled through the slide glass by total internal reflection could produce scattered light in the presence of AuNPs on the slide glass. This allows us to use a 10x objective lens and a USB camera to detect over 10,000 particles in a single image with a large field of view (1 ,248 pm x 702 pm). Using the DF imaging system, we detected target miR-21 -5p miRNAs by quantifying the number of released AuNPs (FIG. 1G). The developed DF imaging device showed promising features for practical applications, providing stable and reliable signal detection without the need for extensive calibration.

[0124] Evaluation of the CRISPR / Cas13a-assisted MGNP-DF assay

[0125] 1) Background noise reduction:

[0126] To enhance the performance of our miRNA detection system, it was essential to minimize background noise in DF imaging, especially for an affordable, portable system. We thus explored the efficacy of black PDMS chambers to improve the signal-to-noise ratio in detecting scattering light from AuNPs. Given the nature of side illumination, background noise variability was anticipated based on the optical properties of the chamber. Comparative analysis between transparent PDMS (PDMS, Sylgard 184) and black PDMS (mixed PDMS with black ink, MG chemicals total ground carbon conductive coating, 838AR) demonstrated the superior noise reduction capabilities of black PDMS, resulting in a twofold improvement in contrast (FIG. 6A).

[0127] 2) Size of magnetic beads and AuNPs:

[0128] The sizes of both the magnetic beads and AuNPs were critical parameters in optimizing the CRISPR / Cas13a-assisted MGNP-DF assay. Initial comparisons between 200 nm- and 1 pm-sized magnetic beads revealed that MGNPs synthesized with 200 nm magnetic beads released approximately twice as many AuNPs post-CRISPR / Cas13a reaction (FIG. 3B). Similarly, different sizes of AuNPs were evaluated. AuNPs smaller than 50 nm were inadequately detected in the portable DF imaging system, showing a minimum size threshold (FIG. 4). Comparative analysis between 60 nm and 80 nm AuNPs demonstrated significant signal differentiation with 60 nm AuNPs depending on Cas13a activity, likely due to different efficiencies of cleavages and AuNP release. Based on these results, we chose 200 nm magnetic beads and 60 nm AuNPs for MGNP synthesis (FIG. 3C).

[0129] 3) Bridge design:

[0130] Three distinct designs of bridge linkers were evaluated for their efficacy in facilitating conjugation between MNPs and AuNPs (FIGS. 3D and 5) and cleavage by CRISPR / Cas13a. In the first design, we employed direct conjugation of AuNPs and magnetic beads through a single-stranded DNA / RNA / DNA linker with biotin and thiol functionalization for its simplicity. However, this design failed to exhibit cleavage by activated CRISPR / Cas13a proteins, rendering it unsuitable for the MGNP-DF assay. In the subsequent designs, we connected AuNPs and magnetic beads by sandwich hybridization with a singlestranded DNA / RNA / DNA linker (FIG. 1A). The DNA / RNA / DNA linkers hybridize to the DNAs functionalized on AuNPs and magnetic beads, respectively. Evaluation post-CRISPR / Cas13a cleavage showed the effectiveness of poly-T sequences located on either side of the RNA sequence, enhancing bridge recognition by CRISPR / Cas13a (see Table 1 for the tested linker sequences).

[0131] Table 1. Sequences of oligomers used in this study

[0132] 4) Reaction time:

[0133] Determining the optimal reaction time for CRISPR / Cas13a was crucial for assay efficiency. Evaluation across reaction times ranging from 1 to 30 minutes revealed rapid AuNP detachment within the first 5 minutes, followed by saturation after 10 minutes. Consequently, 10 minutes was chosen as the optimized reaction time for CRISPR / Cas13a (FIG. 3E).

[0134] Evaluation of detection sensitivity and specificity

[0135] The sensitivity of the CRISPR / Cas13a-assisted MGNP-DF assay was evaluated through serial dilutions of miR-21 -5p, employing MGNPs synthesized under optimized conditions (FIG. 6A). Titration experiments showed a LOD of 500 attomoles for miR-21 -5p, as calculated by the concentration producing a signal equal to mean + 3x standard deviation of the mock sample (FIG. 6B and 7A-B). It should be noted that this is a direct detection of target miRNA without any target sequence amplification (amplification-free detection). Notably, the quantity of released AuNPs from MGNPs exhibited a linear increase corresponding to the miR-21 -5p concentration. To address the specificity of the developed assay, five distinct sequences were additionally employed: wildtype (WT), single-mismatch (SM), and double-mismatches (DM) of miR-21 -5p, miR-421 , and miR-9-5p (sequences were listed in Table 1). As shown in FIG. 6C, DM, miR-421 , and miR-9-5p failed to activate CRISPR / Cas13a and consequently did not induce the release of AuNPs from MGNPs. While the SM sequence exhibited a relatively higher number of released AuNPs compared to other sequences, it also demonstrated a significantly lower release compared to miR-21 -5p (WT). Consequently, the MGNP-DF assay demonstrated the capability to differentiate at least single-base mismatch with high specificity.

[0136] Detection of miR-21-5p from breast cancer cell lines

[0137] The CRISPR / Cas13a-assisted MGNP-DF assay was employed to detect miR-21 -5p levels in five distinct breast cancer cell lines, including HCC1937, HCC1954, MCF7, MDA-MB-231 , and SKBR3 (FIGS. 8A and 8B). We first isolated small RNAs from cell lysates and applied the assay to the isolated small RNAs. Notably, differential expression profiles were observed among the tested cell lines, with HCC1954 and MCF7 exhibiting high miR-21 -5p expression levels, HCC1937 and MDA-MB-231 displaying intermediate expression, and SKBR3 demonstrating low expression (FIG. 8B). To validate the result obtained from our developed system, we performed RT-qPCR, which is considered the gold standard method for miRNA detection. 300 ng of small RNAs extracted from the five breast cancer cell lines were utilized for cDNA synthesis and PCR reactions (FIG. 8C). Notably, our developed system strongly correlated with the RT-qPCR results, as evidenced by a high Pearson correlation coefficient (r = 0.95, p = 0.0123, FIG. 8D). It should also be noted that our MGNP-DF assay required a 20-fold lower amount of RNA as input for detecting miR-21 -5p without target amplification.

[0138] CONCLUSIONS

[0139] The CRISPR / Cas13a-assisted MGNP-DF assay is an approach designed for simple, rapid, and accurate detection of target miRNAs, with a focus on point-of-care operation. The assay employs the CRISPR-Cas13a system for direct miRNA detection, eliminating the need for amplification. This method enables specific recognition of single target miRNAs, providing high sensitivity and specificity.

[0140] In this study, we prepared LwaCas13a and crRNA for miR-21 -5p through bacterial expression and in vitro transcription, respectively. We then synthesized and characterized MGNPs, which are crucial components of the assay. Unlike other detection probes (e.g., fluorophores), nanoparticles exhibit much stronger scattering signals, enabling accurate quantification of individual particles even using a portable system. This leads to high sensitivity without target amplification and accurate quantification, which is important to measure the relative expression of target miRNA between cancers and non-cancer cells. The DF imaging system was compact and efficient, providing stable and reliable signal detection without extensive calibration. The assay was optimized in several aspects, including background noise reduction, size of magnetic beads and AuNPs, bridge design, and reaction time. These optimizations improved the sensitivity and specificity of the assay, allowing for the detection of miR-21 -5p at a LOD of the sub- femtomole range and the differentiation of at least single-base mismatches with high specificity. The validation with multiple breast cancer cell lines and comparison with the gold standard RT-qPCR shows the accuracy of our point-of-care system in quantifying target miRNA levels. We previously demonstrated that miRNA-21 -5p detection in tumor-derived extracellular vesicles accurately identifies ovarian cancer patients from healthy controls. The developed MGNP-DF system could open up the possibility of conducting the test in point-of-care settings in clinics. In conclusion, the CRISPR / Cas13a-assisted MGNP-DF assay is a promising method for miRNA detection, offering simplicity, rapid analysis, accurate quantification, and point-of-care operation. Its high sensitivity, specificity, and reliability make it a valuable tool for miRNA research and clinical applications.

[0141] The above-described results were obtained using the following materials and methods.

[0142] MATERIALS AND METHODS

[0143] Cell culture

[0144] The human breast cancer cell lines, HCC1937, HCC1954, SKBR3, MCF7, and MDA-MB-231 , were purchased from American Type Culture Collection (ATCC). HCC1937, HCC1954, and SKBR3 cells were cultured in RPMI-1640 (Cytiva), and MCF7 and MDA-MB-231 were maintained in DMEM (Cytiva) at 37 °C in 5% CO2. All basal media were supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 pg / mL streptomycin (Millipore Sigma).

[0145] Purification of LwaCas13a

[0146] We performed a purification of LwaCas13a as previously reported.28The plasmid DNA encoding LwaCas13a from Feng Zhang group (Addgene plasmid #90097;n2t.net / addgene:90097; RRID:Addgene_90097) were transformed into E.coli strain [Rosetta 2(DE3) pLysS (Millipore Sigma)] for bacterial expression. The LwaCas13a are purified by following steps of cell lysis, 1 st Ni-NTA purification, SUMO protease (ThermoFisher Scientific) treatment, and 2nd NI-NTA purification (FIG. 1B). The purified LwaCas13a were stored in the buffer (50 mM Tris-HCI, pH 7.5, 600 mM NaCI, and 2 mM DTT) with 5% glycerol and protease inhibitor until use at -80°C. crRNA generation

[0147] The crRNA for detecting miR-21 -5p was generated by in vitro transcription. Briefly, the universal upper strand DNA, including the T7 promoter sequence, was annealed with the bottom strand DNA, including reverse complement T7 promoter and crRNA sequences. In vitro transcription was conducted with the annealed DNAs according to the manufacturer’s description (Promega) and confirmed by 15% denaturing polyacrylamide gel electrophoresis (FIG. 1C). All DNA sequences used for crRNA generation are shown in Table 1.

[0148] Quantitative RT-qPCR

[0149] Small RNA isolation and RT-qPCR were performed as previously described.28Briefly, small RNAs from the five different cells were isolated using the differential ethanol precipitation method. A total of 300 ng of small RNAs from HCC1937, HCC1954, MDA-MB-231 , MCF7, and SKBR3 were used following polyadenylation and reverse transcription. The quantitative PCR was conducted using CFX Opus Real-Time PCR systems (Biorad), and the relative miRNA levels were determined using the -ACq values. U6 snRNA levels were used as an internal control. All DNA sequences used for RT-qPCR are shown in Table 1.

[0150] Preparation of magnetic beads with linkers

[0151] 200 nm streptavidin-coated hydrophilic magnetic beads were purchased from Ocean Nanotech (SV002000, 1 mg / mL). We washed magnetic beads twice with a binding / wash buffer (pH = 7.5, 5 mM Tris-HCI, 1 M NaCI, and 0.5 mM EDTA). We added biotinylated MNP-linker (10 pM, 5 pL) and incubated the mixture for 2 hours at RT on a Hula mixer, followed by five-time washes with a wash buffer A (pH = 7.2, 20 mM HEPES, and 0.01 % Tween 20) by magnetic separation to remove unbound MNP-linker. MNPs conjugated with MNP-linker were stored in a storage buffer (pH = 7.2, 20 mM HEPES, 0.1 % BSA, and 0.01 % Tween 20) at 4°C. All linker sequences used for MNP conjugation are listed in Table 1 .

[0152] Preparation of gold nanoparticles (AuNPs) with linkers

[0153] We used the previously described method to conjugate AuNP-linker to AuNPs.30We mixed unconjugated 60 nm AuNPs (TedPella, 15709-20, 5.2 x 1011particles / mL; 50 pL), 50 mM HEPES 40 pL, and 200 pM BSA 10 |al_, and incubated the mixture for 30 min at RT on a Hula mixer, followed by threetime washes with a wash buffer A (pH = 7.2, 20 mM HEPES, and 0.01% Tween-20) via centrifugation (3,500 X g, 5 min). After washing, we added the TCEP-treated AuNP-linker (10 pM, 10 pL) into AuNP solution containing 300 mM KCI and incubated the mixture for 3 hours at RT on a Hula mixer. The AuNPs were washed with a wash buffer A (pH = 7.2, 20 mM HEPES, and 0.01% Tween-20) via centrifugation (3,500 X g, 5 min, 3 times). AuNPs conjugated with AuNP-linker were stored in a storage buffer (pH = 7.2, 20 mM HEPES, 0.1% BSA, and 0.01% Tween-20) at 4°C. All linker sequences used for AuNP conjugation are listed in Table 1.

[0154] Preparation of magnetic gold nanoparticles (MGNPs)

[0155] We prepared magnetic gold nanoparticles using 200 nm magnetic beads and 60 nm AuNPs connected with DNA / RNA / DNA bridges. We first added the DNA linker-conjugated magnetic beads (1 mg / mL; 10 pL) and the DNA / RNA / DNA bridge (25 nM, 4 pL) in a hybridization buffer (pH = 7.2, 20 mM HEPES, 200 mM KCI, 0.1% BSA, and 0.01% Tween-20). We incubated the mixture for 30 min at 37°C and then washed the magnetic beads five times by magnetic separation with a wash buffer B (pH = 7.2, 20 mM HEPES, 200 mM KCI, and 0.01% Tween-20) to remove the unbound bridge. The washed magnetic beads were resuspended in a hybridization buffer (pH = 7.2, 20 mM HEPES, 200 mM KCI, 0.1% BSA, and 0.01% Tween-20). We added the AuNPs conjugated with AuNP-linker (2.6 x 1011particles / mL; 20 pL) to the magnetic bead solution and incubated the mixture for 30 min at 37°C. The MGNPs were formed after the incubation. The unbound AuNPs were removed by five-time washes with a wash buffer C (pH = 7.2, 20 mM HEPES, 5 mM KCI, and 0.01% Tween-20) by magnetic separation. The washed MGNPs were stored overnight at 4°C on a Hula mixer before use.

[0156] Preparation of a substrate coated with capture linkers

[0157] A black PDMS well was attached to a glass slide. We introduced 5 pL of streptavidin solution (Millipore Sigma, 50 pg / mL in 1 x PBS) into the PDMS well and allowed it to incubate for 2 hours at RT. Following incubation, the PDMS well was rinsed with a binding / wash buffer (pH = 7.5, 5 mM Tris-HCI, 1 M NaCI, and 0.5 mM EDTA). Upon completion of the streptavidin coating, capture linkers (500 pM, dissolved in the binding / wash buffer) were added to the PDMS well and incubated for 1 hour at RT. After incubation, unbound capture linkers were removed with the binding / wash buffer. The substrate coated with capture linkers was then stored at 4°C.

[0158] CRISPR / Cas13a-assisted MGNP-DF assay

[0159] First, LwaCas13a (400 nM) and crRNA (200 nM) were mixed and incubated at RT for 15 min. We then mixed the aforementioned concentration of miR-21 -5p or 15 ng of small RNA fraction from cell lines, Cas13a / crRNA complex, MGNPs, and reaction buffer (pH = 7.2, 20 mM HEPES, 60 mM KCI, 6 mM MgCI2, 0.1% BSA, and 0.05% Tween-20) with a final volume of 20 pL, and incubated at RT for 10 min on a Hula mixer. After 10 min incubation, the MGNPs were separated by a magnet. The supernatant was added to a black PDMS well attached to a slide glass and incubated for 15 min at 50°C. The AuNPs contained in the supernatant were conjugated to the capture linkers coated on the slide glass. After 15 min incubation, we washed the black PDMS well with a wash buffer D (pH = 7.2, 20 mM HEPES, 60 mM KCI, 6 mM MgCl2, and 0.01% Tween-20) to remove the unbound AuNPs. The washed well was covered by a cover glass, and images for AuNPs were obtained using the portable dark-field microscope.

[0160] Image analysis

[0161] The quantification of AuNPs was conducted using the Imaged Comet plugin, with consistent detection parameters applied across all samples (approximate particle size: 4.0 pixels, intensity threshold: 3.0).

[0162] Statistical analysis

[0163] All data were analyzed with GraphPad Prism (Version 10, GraphPad Software Inc., San Diego, CA, USA) and displayed as mean ±standard deviation. The unpaired t-test and two-way ANOVA with Boneferroni’s multiple comparison tests were used to compare with control sets. Statistical significance was considered for values of p < 0.05.

[0164] OTHER EMBODIMENTS

[0165] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0166] Other embodiments are within the claims.

Claims

CLAIMS1 . A method of detecting a microRNA (miRNA) comprising:(a) contacting a sample comprising the miRNA with:(i) a first particle conjugated to a first polynucleotide;(ii) a second particle conjugated to a second polynucleotide; and(iii) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide;(b) contacting the sample with a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and a CRISPR associated (CAS) protein;(c) allowing the crRNA to hybridize to the miRNA, thereby activating the CAS protein to cleave the bridge polynucleotide; and(d) detecting the first particle that is conjugated to the first polynucleotide.

2. The method of claim 1 , wherein the first polynucleotide and second polynucleotide are a single polynucleotide integral with the bridge polynucleotide.

3. The method of claim 1 , wherein the first polynucleotide and the second polynucleotide are separate polynucleotides, and the bridge polynucleotide comprises a first portion that hybridizes to the first polynucleotide and a second portion that hybridizes to the second polynucleotide.

4. The method of any one of claims 1 -3, wherein the bridge polynucleotide comprises a target cleavage sequence that is cleaved by the CAS protein.

5. The method of claim 4, wherein the bridge polynucleotide comprises a spacer region 5’ and / or 3’ of the target cleavage sequence.

6. The method of claim 5, wherein the spacer region comprises a polyT sequence.

7. The method of any one of claims 1 -6, further comprising, following step (c), separating the first particle from the sample.

8. The method of claim 7, wherein separating comprises immobilizing the first particle.

9. The method of claim 8, wherein the method comprises immobilizing the first particle with a capture agent that binds to the first polynucleotide.

10. The method of claim 9, wherein the capture agent is conjugated to a glass slide.11 . The method of claim 10, wherein the capture agent is biotinylated, and the glass slide is coated with streptavidin.

12. The method of any one of claims 9-11 , wherein the capture agent is a third polynucleotide that hybridizes to the first polynucleotide.

13. The method of any one of claims 9-12, wherein the method comprises incubating the sample with the capture agent for 1 to 30 minutes.

14. The method of claim 13, wherein the method comprises incubating the sample with the capture agent for 10 to 15 minutes.

15. The method of any one of claims 1 -14, wherein the first particle comprises a plurality of the first polynucleotides conjugated to the first particle.

16. The method of any one of claims 1 -15, wherein the first particle is a metallic nanoparticle.

17. The method of claim 16, wherein the metallic nanoparticle is a gold nanoparticle.

18. The method of claim 17, wherein the gold nanoparticle is from 10 nm to 200 nm in diameter.

19. The method of claim 18, wherein the gold nanoparticle is 60 nm in diameter.

20. The method of any one of claims 1 -19, wherein the first polynucleotide comprises a thiol.21 . The method of claim 20, wherein the first particle is treated with tris(2-carboxyethyl)phosphine (TCEP) and conjugated to the thiol of the first polynucleotide.

22. The method of any one of claims 1 -21 , further comprising, following step (c), separating the second particle from the sample.

23. The method of claim 22, wherein separating comprises immobilizing the second particle.

24. The method of claim 23, wherein the second particle is a magnetic bead.

25. The method of claim 24, wherein the magnetic bead is coated with streptavidin.

26. The method of claim 25, wherein the second polynucleotide is biotinylated.

27. The method of any one of claims 24-26, wherein the separating comprises using a magnet to immobilize the magnetic bead.

28. The method of any one of claims 24-27, wherein the magnetic bead is from 100 nm to 500 nm in diameter.

29. The method of claim 28, wherein the magnetic bead is 200 nm in diameter.

30. The method of any one of claims 24-29, wherein the method comprises separating the second particle from the sample prior to separating the first particle from the sample.31 . The method of any one of claims 24-30, wherein the method comprises separating the second particle from the sample and saving a supernatant comprising the first particle.

32. The method of any one of claims 1 -31 , wherein the second particle comprises a plurality of the second polynucleotides conjugated to the second particle.

33. The method of any one of claims 1 -32, wherein the sample comprises a plurality of the first particles and a plurality of the second particles.

34. The method of any one of claims 1 -33, wherein step (d) comprises detecting the first particle with dark-field imaging.

35. The method of claim 1 -34, wherein the step (d) comprises side illumination of a light-emitting diode (LED) light.

36. The method of claim 35, wherein step (d) comprises side illumination light coupled with total internal reflection to produce scattered light.

37. The method of any one of claims 1 -36, wherein step (d) comprises detecting the first particle for 1 to 5 minutes.

38. The method of claim 37, wherein step (d) comprises detecting the first particle for 3 minutes.

39. The method of any one of claims 1 -38, wherein the CAS protein is CAS13a.

40. The method of any one of claims 1 -39, wherein the miRNA is miR-21 -5p, miR-9-5p, or miR-421 .41 . The method of any one of claims 1 -40, wherein the first polynucleotide, the second polynucleotide, and / or the bridge polynucleotide is a deoxyribonucleotide (DNA), a ribonucleotide (RNA), or a combination thereof.

42. The method of claim 41 , wherein the first polynucleotide is DNA, the second polynucleotide is DNA, and the bridge polynucleotide is RNA.

43. The method of any one of claims 1 -42, further comprising, prior to step (c), forming a complex with the crRNA and the CAS protein.

44. The method of any one of claims 1 -43, wherein a level of expression of the miRNA in the sample is associated with cancer, and the method comprises diagnosing a subject with cancer upon detection of the miRNA above a predetermined threshold in the sample.

45. The method of claim 43, wherein the cancer is breast cancer or ovarian cancer.

46. The method of claim 43 or 44, further comprising treating the cancer.

47. A system or kit for detecting miRNA comprising one or more of:(a) a first particle conjugated to a first polynucleotide;(b) a second particle conjugated to a second polynucleotide;(c) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide;(d) a crRNA;(e) a CAS protein, wherein the miRNA is configured to hybridize to the crRNA to activate the CAS protein to cleave the bridge polynucleotide; and(f) a dark field microscope.

48. A composition comprising(i) a first particle conjugated to a first polynucleotide;(ii) a second particle conjugated to a second polynucleotide; and(iii) a bridge polynucleotide that connects the first polynucleotide to the second polynucleotide.

49. The method of claim 48, wherein the first polynucleotide and second polynucleotide are a single polynucleotide integral with the bridge polynucleotide.

50. The method of claim 48, wherein the first polynucleotide and the second polynucleotide are separate polynucleotides, and the bridge polynucleotide comprises a first portion that hybridizes to the first polynucleotide and a second portion that hybridizes to the second polynucleotide.51 . The composition of any one of claims 48-50, further comprising a miRNA.

52. The composition of any one of claims 48-51 , further comprising a crRNA and a CAS protein, wherein the CAS protein is configured to cleave the bridge polynucleotide.

53. The composition of any one of claims 48-52, wherein the first particle is a magnetic bead.

54. The composition of any one of claims 47-53, wherein the second particle is a gold nanoparticle.

55. A composition comprising:(i) a metal particle conjugated to a first polynucleotide; and(ii) a substrate conjugated to a capture agent, wherein the capture agent is attached to the first polynucleotide.

56. The composition of claim 55, wherein the capture agent is a second polynucleotide that is hybridized to the first polynucleotide.

57. The composition of claim 55 or 56, wherein the metal particle comprises a plurality of the first polynucleotides.

58. The composition of claim 57, wherein at least one of the plurality of first polynucleotides is hybridized to a portion of a bridge polynucleotide cleaved by a CAS protein.

59. The composition of any one of claims 55-58, wherein the metal particle is a gold nanoparticle.

60. The composition of any one of claims 55-59, wherein the substrate is a glass slide.61 . The composition of claim 60, wherein the capture agent is biotinylated, and the glass slide is coated with streptavidin.

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