Nucleic acid amplification circuit-based hydrogel assay for one-step detection of exosomal mirna

Abstract

The present invention relates to a composition for detecting a target nucleic acid using a hydrogel and a rolling circle amplification (RCA) reaction, and a use thereof. The composition for detecting a target nucleic acid according to the present invention is capable of rapidly detecting a target nucleic acid even at a very low concentration, and is thus suitable for high-sensitivity on-site detection. Furthermore, the composition has been confirmed to exhibit excellent detection performance for a target nucleic acid even in preclinical and clinical samples, and is thus useful for cancer diagnosis.

Description

Nucleic acid amplification circuit-based hydrogel assay for one-step detection of exosomal MIRNA

[0001] The present invention relates to a composition for detecting target nucleic acids using a hydrogel and a rolling circle amplification (RCA) reaction, and to the use thereof.

[0002] When nucleic acids are extracted from biological samples, such as blood, for analysis, the extracted amount is generally too small to be used directly for various analyses; therefore, amplification of the extracted nucleic acids is necessary for accurate analysis. Various nucleic acid amplification technologies have been developed to date. In particular, PCR (Polymerase Chain Reaction), a representative method for DNA amplification, is widely used in various fields because it is a highly efficient amplification technique that selectively amplifies target genes in large quantities. However, PCR has disadvantages such as high costs and difficulty in use for point-of-care diagnostics, as it requires a PCR instrument capable of precise temperature control of the reaction solution. Furthermore, in genetic diagnosis, accuracy and sensitivity are often unsuitable due to issues such as non-specific reactions caused by an excessive amount of initiator at the beginning of the reaction and reduced amplification efficiency resulting from polymer recombination in the later stages.

[0003] Against this backdrop, research and development on new diagnostic platforms with enhanced sensitivity are underway, and recently, Rolling Circle Amplification (RCA) reactions are being utilized in various fields. Based on circular template DNA, the RCA reaction can amplify minute amounts of genes at room temperature (25±5℃) without temperature fluctuations. However, it involves the inconvenience of having to go through two steps: a ligation step in which the two ends of a linear padlock probe are joined in a circular shape, and an amplification step in which the gene is amplified in a circular fashion using short-length primers.

[0004] Accordingly, the inventors designed a new nucleic acid detection composition capable of detecting a target nucleic acid in one step using a hydrogel comprising a hybridized target nucleic acid, circular DNA, and a capture probe, and completed the present invention by confirming that the composition has excellent nucleic acid detection capabilities.

[0005] One object of the present invention is to provide a composition for detecting a target nucleic acid comprising a hydrogel comprising: (i) a capture probe having a sequence complementary to a portion of the target nucleic acid and having its ends immobilized on the hydrogel; (ii) circular DNA having a sequence complementary to a portion of the target nucleic acid; and (iii) a reporter probe that binds to an amplification product.

[0006] Another objective of the present invention is to provide a method for detecting a target nucleic acid using the composition for detecting the target nucleic acid.

[0007] Another objective of the present invention is to provide a method for providing information about cancer using the composition for detecting the target nucleic acid.

[0008] In the following, redundant details will be omitted to prevent clutter. In other words, the content of the invention is not limited solely to the following, and should be interpreted in accordance with the overall context of the invention.

[0009] The present invention will be described in detail below.

[0010] The present invention provides a composition for detecting a target nucleic acid comprising a hydrogel comprising: (i) a capture probe having a sequence complementary to a portion of the target nucleic acid and having its ends immobilized on the hydrogel; (ii) circular DNA having a sequence complementary to a portion of the target nucleic acid; and (iii) a reporter probe that binds to an amplification product.

[0011] The composition for detecting target nucleic acids according to the present invention is an enzymatic cascade reaction system utilizing a rolling circle amplification (RCA) reaction, which facilitates one-step detection by hybridizing target nucleic acids, circular DNA, and capture probes into a hydrogel, and enables stable detection of target nucleic acids by exponentially amplifying them.

[0012] According to one embodiment, the composition for detecting target nucleic acids of the present invention is referred to as a nucleic acid amplification circuit-based hydrogel assay (NACH assay, NACH analysis) system.

[0013] Specifically, as shown in FIGS. 1(a) and 1(b), the nucleic acid detection composition of the present invention has a portion of the target nucleic acid bound to a capture probe immobilized on a hydrogel, and another portion bound to circular DNA inside the hydrogel. Subsequently, a rolling circle amplification (RCA) reaction for the target nucleic acid is initiated by a nucleic acid polymerase. A reporter probe binds to the amplification product of the target nucleic acid generated by the RCA reaction, and the target nucleic acid can be detected by emitting a signal (e.g., a fluorescent signal) detectable by the reporter probe after a washing process of the hydrogel.

[0014] In the present invention, "hydrogel" refers to a material formed when an organic polymer (natural or synthetic) is cross-linked through covalent, ionic, or hydrogen bonds to capture water molecules and create a three-dimensional open lattice structure that forms a gel. The hydrogel is characterized by comprising a target nucleic acid, circular DNA, and a capture probe hybridized together.

[0015] Hydrogel particles may comprise hydrophilic monomers or polymers. In another aspect of the present invention, hydrogel particles may comprise one or more selected from the group consisting of natural polymers, acrylic monomers or polymers, polyacrylamide monomers or polymers, phosphatidyl choline, hyaluronic acid monomers or polymers, carboxymethyl cellulose, alginate, chitosan, poly(e-caprolactone), poly(lactic acid), poly(glycolic acid), polyethylene glycol, hydroxyapatite, tricalcium phosphate, and mixtures thereof.

[0016] The natural polymer comprises one or more selected from the group consisting of red algae-derived polysaccharides such as carrageenan, agar or agarose, mannose-containing polysaccharides such as mannan, galactomannan, glucomannan or derivatives thereof, and natural gums such as locust bean gum, guar gum, xanthan gum, arabic gum, gellan gum, or karaya gum.

[0017] The acrylic monomer or polymer includes a hydrophilic acrylic monomer or polymer, specifically comprising one or more selected from the group consisting of polyethylene glycol diacrylate, polyethylene glycol methacrylate, polymethyl methacrylate (PMMA), hydroxyethyl acrylate (HEA), and hydroxyethyl methacrylate (HEMA).

[0018] In another aspect of the present invention, hydrogel particles may preferably comprise an acrylic monomer or polymer capable of radical polymerization to ensure wide usability, and specifically, may preferably comprise a polyethylene glycol acrylate monomer or polymer.

[0019] More preferably, a mixture of polyethylene glycol and a polyacrylamide-based monomer or polymer may be used. More specifically, it may include polyethylene glycol and one or more selected from the group consisting of polyethylene glycol diacrylate, polyethylene glycol methacrylate, polymethyl methacrylate (PMMA), hydroxyethyl acrylate (HEA), and hydroxyethyl methacrylate (HEMA); and more specifically, a mixture of polyethylene glycol and polyethylene glycol diacrylate may be used. That is, it may include a mixture of polyethylene glycol and polyethylene glycol diacrylate.

[0020] In this invention, the term "hybridization" refers to the formation of double-stranded nucleic acids through hydrogen bonding between single-stranded nucleic acids having complementary base sequences, and is used with a meaning similar to annealing. However, in a slightly broader sense, hybridization includes not only cases where the base sequences of two single strands are perfectly complementary (perfect match), but also exceptional cases where some base sequences are not complementary (mismatch).

[0021] According to one embodiment, the hydrogel may be prepared by pouring a hydrogel precursor solution (which may include PEG-DA, PEG, TE buffer and HMPP) into a hydrogel mold prepared using soft lithography and photopolymerizing it.

[0022] In the present invention, "rolling circle amplification (RCA) reaction" refers to a nucleic acid amplification reaction that amplifies a circular nucleic acid template (e.g., a single-stranded DNA loop) through a rolling circle mechanism. A rolling circle amplification reaction is initiated by the hybridization of a primer to a circular, often single-stranded, nucleic acid template. Then, a nucleic acid polymerase proceeds around the circular nucleic acid template to expand the primer hybridized to the circular nucleic acid template and repeat the sequence of the nucleic acid template (rolling circle mechanism). Rolling circle amplification typically produces a concatemer containing tandem repeat units of the circular nucleic acid template sequence. Rolling circle amplification may be a linear RCA (LRCA) exhibiting linear amplification kinetics (e.g., RCA using a single specific primer) or an exponential RCA (ERCA) exhibiting exponential amplification kinetics. Rolling circle amplification may also be performed using multiple primers to generate over-branched concatemers (multiple-primed rolling circle amplification or MPRCA). For example, in double-primed RCA, one primer may be complementary to the circular nucleic acid template, as in linear RCA, while the other primer may be complementary to the tandem repeat unit nucleic acid sequence of the RCA product. Consequently, double-primed RCA can proceed with a chain reaction having exponential (geometric) amplification kinetics, characterized by a branching cascade of multiple hybridization, primer extension, and strand separation phenomena involving both primers. This commonly produces a distinct set of concatemer-type double-stranded nucleic acid amplification products. Rolling circle amplification can be performed in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0023] Accordingly, the composition for detecting target nucleic acids according to the present invention may further include dNTPs and nucleic acid polymerases for an RCA reaction.

[0024] The above dNTPs (deoxynucleotide triphosphates) include dATP, dTTP, dGTP, and dCTP, and play a role in extending the DNA sequence.

[0025] The above nucleic acid polymerase is an enzyme that plays a role in initiating the RCA reaction, and may be any one selected from the group consisting of Phi29 DNA polymerase, phage M2 DNA polymerase, phage Phi-PRD1 DNA polymerase, VENT.RTM DNA polymerase, Klenow fragment DNA polymerase I, T5 DNA polymerase, PRD1 DNA polymerase, T4 DNA polymerase holoenzyme, T7 native polymerase, and Bst polymerase, and preferably may be Phi29 DNA polymerase.

[0026] In the present invention, "target nucleic acid" refers to a nucleic acid sequence to be detected and is used interchangeably with "target nucleic acid" or "target sequence," etc. For example, it may be miRNA, miRNA sponge, tough decoy miRNA, anti-miR, small RNA, siRNA, shRNA, or any combination thereof.

[0027] In the present invention, the term "capture probe" may be used interchangeably with "binding probe," "capture probe," etc., and is characterized by containing a sequence complementary to a portion of the target nucleic acid to be detected and having its terminals immobilized on a hydrogel. In the present invention, the term "complementary" refers to the ability to form precise pairs between two nucleotides. That is, if a nucleotide at a given position of a nucleic acid can form hydrogen bonds with a nucleotide of another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. Complementaryness between two single-stranded nucleic acid molecules may be "partial" when only a portion of the nucleotides bind, or complete when total complementaryness exists between the single-stranded molecules. The degree of complementaryness between nucleic acid strands significantly affects the efficiency and strength of hybridization between the nucleic acid strands.

[0028] According to one embodiment, the capture probe may have one or more groups selected from the group consisting of acrylamide, glutaraldehydes, and adipic acid dihydrazide attached to its 3' end. More specifically, having an acrylamide group attached, it may be immobilized within the hydrogel by interacting with polyethylene glycol diacrylate (PEG-DA), which is a hydrogel precursor.

[0029] In the present invention, "circular DNA" refers to a DNA molecule in a circular form and is characterized by including a sequence complementary to a portion of the target nucleic acid to be detected, excluding the sequence that binds complementarily to the capture probe.

[0030] According to one embodiment, the circular DNA can be formed through the circularization of a padlock probe via phosphodiester bonding.

[0031] In the present invention, a "reporter probe" is characterized by comprising a substance (e.g., a fluorescent label) that binds to the amplification product of a target nucleic acid generated by an RCA reaction and emits a detectable signal. In the present invention, a "detectable signal" refers to a signal that can be directly detected by the human eye or by means of a detection system. The characteristics of said signal vary depending on the characteristics of the label used. The signal may be, in particular, a colored, luminescent, fluorescent, phosphorescent, radioactive, or magnetic signal. Preferably, said signal is a fluorescent signal.

[0032] The reporter probe described above independently comprises a fluorescent labeling substance and can exhibit a fluorescent signal by binding to the amplification product of the target nucleic acid. The reporter probe may include, for example, any one selected from the group consisting of ALEX-350, FAM, VIC, TET, CAL Fluor®Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, and Quasar 705, and preferably may include FAM or CY3.

[0033] According to one embodiment, depending on the type of miRNA, the reporter probe used for detection may be 5' labeled with Fluorescein amidite (FAM), and the reporter probe may be 5' labeled with Cyanine3 (Cy3). These fluorescent labels may be applied differently depending on the technical content.

[0034] The composition for detecting target nucleic acids according to the present invention can be optimized when the concentration of dNTPs is 300 to 500 μM (preferably 400 μM), the concentration of Phi29 DNA polymerase is 8 to 12 units (preferably 10 units), and the concentration of reporter probe is 500 to 1000 nM.

[0035] According to one embodiment, the concentration of dNTPs is optimized when the concentration of Phi29 DNA polymerase is 400 μM, the concentration of Phi29 DNA polymerase is 10 units, and the concentration of reporter probe is 500 to 1000 nM, and under these conditions, the composition for detecting target nucleic acids of the present invention exhibits the highest detection efficiency.

[0036] In addition, the present invention provides a method for detecting a target nucleic acid using the above-described composition for detecting a target nucleic acid.

[0037] Specifically, the target nucleic acid detection method of the present invention comprises the following steps:

[0038] (a) a step of treating a sample on a hydrogel comprising: (i) a capture probe having a sequence complementary to a portion of the target nucleic acid and having its ends immobilized on the hydrogel; (ii) circular DNA having a sequence complementary to a portion of the target nucleic acid; and (iii) a reporter probe that binds to the amplification product;

[0039] (b) a step in which the target nucleic acid of the above sample is amplified; and

[0040] (c) A step in which a reporter probe binds to the target nucleic acid amplified in step (b) above to generate a detectable signal.

[0041] Step (b) above may involve amplifying the target nucleic acid of the sample through a rolling circle amplification (RCA) reaction. Accordingly, it may include dNTPs and nucleic acid polymerase for the RCA reaction.

[0042] The detectable signal in step (c) above may be a fluorescent signal emitted by a reporter probe containing a fluorescent labeling substance on the target nucleic acid.

[0043] In the method for detecting a target nucleic acid of the present invention, "sample" means a biological sample containing any nucleic acid, for example, RNA. For example, it may be miRNA, miRNA sponge, turf decoy miRNA, anti-miR, small RNA, siRNA, shRNA, or any combination thereof.

[0044] The above biological sample may be any tissue or body fluid obtained from the subject.

[0045] The biological sample comprises, but is not limited to, the subject's sputum, blood, serum, plasma, blood cells (e.g., leukocytes), tissue, biopsy sample, smear sample, wash sample, swab sample, cell-containing body fluid, liquid nucleic acid, urine, peritoneal fluid and pleural fluid, cerebrospinal fluid, feces, leaky fluid, or cells derived therefrom. The biological sample may also comprise tissue sections taken for histological purposes, i.e., frozen or fixed sections, or microscopic cells or extracellular portions thereof. The biological sample may be obtained in a manner that does not cause harm to the subject.

[0046] In addition, the present invention provides a method for providing information about cancer using the above-mentioned composition for detecting target nucleic acids.

[0047] Specifically, the method for providing information about cancer according to the present invention comprises the following steps:

[0048] (a) a step of treating a sample on a hydrogel comprising: (i) a capture probe having a sequence complementary to a portion of the target nucleic acid and having its ends immobilized on the hydrogel; (ii) circular DNA having a sequence complementary to a portion of the target nucleic acid; and (iii) a reporter probe that binds to the amplification product;

[0049] (b) a step in which the target nucleic acid of the above sample is amplified; and

[0050] (c) A step in which a reporter probe binds to the target nucleic acid amplified in step (b) above to generate a detectable signal.

[0051] Specifically, a step of treating a sample on a hydrogel comprising: (a) (i) a capture probe having a sequence complementary to a portion of the miRNA and having its ends immobilized on the hydrogel; (ii) circular DNA having a sequence complementary to a portion of the miRNA; and (iii) a reporter probe that binds to the amplification product;

[0052] (b) a step in which the miRNA of the above sample is amplified; and

[0053] (c) A method for providing information about cancer, comprising the step of binding a reporter probe to the miRNA amplified in step (b) to generate a detectable signal.

[0054] The relationship between cancer and miRNA is well-documented in numerous studies. miRNA has emerged as an important novel class of regulatory RNAs that exert profound influence on a wide range of biological processes. These small, non-coding RNA molecules can regulate protein expression patterns through the promotion of RNA degradation, inhibition of mRNA translation, and influence on gene transcription. miRNA plays a central role in various processes, including development and differentiation, control of cell proliferation, stress responses, and metabolism. Accordingly, the expression of numerous miRNAs has been found to be altered in many types of human cancer, and strong evidence has been presented to support the hypothesis that, in some cases, such alterations may play a causative role in tumor progression. Considering this, cancer can be diagnosed by designing and utilizing the above compositions for miRNAs that are commonly known to be associated with cancer.

[0055] The above cancer may be any one selected from the group consisting of stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, and gallbladder cancer, and preferably may be stomach cancer.

[0056] The composition for detecting target nucleic acids according to the present invention is suitable for high-sensitivity on-site detection as it enables rapid detection of target nucleic acids even at very low concentrations, and is useful for cancer diagnosis as it has been confirmed to exhibit excellent detection performance for target nucleic acids in preclinical and clinical samples.

[0057] Figure 1 is a diagram regarding the design of a nucleic acid amplification circuit-based hydrogel (NACH) analysis system.

[0058] (a) This is an overall schematic diagram of a method for detecting exosomal miRNAs that induce EMT promotion in gastric cancer patients.

[0059] (b) This is a diagram schematically illustrating the method of detecting exosomal miRNA using the NACH analysis system.

[0060] Figure 2 is a diagram of the mechanism of functioning of the NACH analysis system.

[0061] (a) Left: A diagram showing a schematic representation of circular DNA synthesis.

[0062] Right side of (a): A figure showing the PAGE analysis results of circular DNA and padlock probes. (1, 2: padlock probe and circular DNA of miRNA-21, respectively; 3, 4: padlock probe and circular DNA of miRNA-99a, respectively)

[0063] (b,d) Figure showing the fluorescence intensity associated with nucleic acid amplification circuits in solution containing (b) miRNA-21 and (d) miRNA-99a at various concentrations.

[0064] (c,e) Figures showing the PAGE analysis results of amplification products generated using various concentrations of (c) miRNA-21 and (e) miRNA-99a. (1, 4: 1 nM; 2, 5: 10 nM; 3, 6: 100 nM; 1, 2, 3: GelRed staining; 4, 5, 6: SYBR Gree II staining)

[0065] (f) This is a diagram showing a schematic representation of the hydrogel manufacturing process.

[0066] (g,h) This figure confirms the optimal conditions of the NACH analysis system using reporter probes of various concentrations (g: miRNA-21; h: miRNA-99a).

[0067] Each of the above data represents the mean ± standard deviation (SD) of three independent experiments (***p < 0.0005, **p < 0.005).

[0068] Figure 3 is a figure confirming the optimization conditions of enzyme concentration in the NACH analysis system.

[0069] (a) This is a diagram confirming the optimal conditions by measuring the fluorescence intensity for dNTPs at various concentrations.

[0070] (b) This figure shows the results of confirming the optimal conditions by measuring the fluorescence intensity of Phi29 DNA polymerase at various concentrations.

[0071] Figure 4 shows the hybridization and amplification products between circular DNA, target miRNA, and capture probe analyzed by PAGE in a NACH analysis system. (1: Binding between circular DNA and capture probe mediated by target miRNAs (miR-21 and miR-99a); 2: Amplification products of miR-21 and miR-99a; 3: Synthetic detection probe and hybridized amplification products)

[0072] Figure 5 shows the spectra obtained by performing Fourier transform infrared spectroscopy (FTIR) on PEG (gray) and PEG-DA (red).

[0073] Figure 6 is a figure confirming that biomarkers (miRNA-21 and miRNA-99a) related to metastatic gastric cancer can be detected through the NACH analysis system.

[0074] (a,b) Figure confirming the sensitivity of the NACH analysis system to (a) miRNA-21 and (b) miRNA-99 at various miRNA concentrations (1 fM to 100 nM).

[0075] (c,d) This figure shows the calibration curves corresponding to the expected limit of detection (LOD) of the NACH assay system for (c) miRNA-21 and (d) miRNA-99a. (LOD = 3 × SD of linear regression (σ) / slope (m))

[0076] (e,f) Figures confirming the selectivity and cross-reactivity of the NACH analysis system for different miRNAs (target miRNA and 1bp and 2bp mismatched miRNAs).

[0077] Each of the above data represents the mean ± standard deviation (SD) for three independent experiments.

[0078] Figure 7 is a figure evaluating the cell RNA detection performance of the NACH analysis system.

[0079] (a,b) Figure showing the results of qRT-PCR analysis on the expression levels of (a) miRNA-21 and (b) miRNA-99a in SNU-484 and Hs 746T gastric cancer cell lines. The expression levels of miRNAs were normalized to the expression levels of miRNA-16 and miRNA-191 (reference genes).

[0080] (c,d) Figure showing the results of measuring the fluorescence images and intensities corresponding to (c) miRNA-21 and (d) miRNA-99a using total cellular RNA (50 ng / hydrogel) isolated from SNU-484 and Hs 746T.

[0081] Each of the above data represents the mean ± SD for three independent experiments (***p < 0.0005, **p < 0.005).

[0082] Figure 8 is a figure confirming the performance of the NACH analysis system on preclinical samples.

[0083] (a) This is a schematic diagram showing the detection of exosomal miRNA using a NACH analysis system in the culture medium of gastric cancer cell lines.

[0084] (b,c) Figure showing the results of analyzing the expression of (b) miRNA-21 and (c) miRNA-99a in the culture medium of gastric cancer cells by qRT-PCR. The expression levels of miRNAs were normalized to the expression levels of miRNA-16 and miRNA-191 (reference genes).

[0085] (d,e) Figures showing the fluorescence images and intensity measurements of (d) miRNA-21 and (e) miRNA-99a when total exosomal miRNA (80 ng / test) isolated from gastric cancer cell culture medium was used (n = 3). The total exosomal miRNA was isolated from the plasma of three different mouse groups (Normal, SNU-484, and Hs 746T).

[0086] (f) This is a schematic diagram showing the strategy used to detect target miRNA in exosomes isolated from the plasma of mice transplanted with gastric cancer cell lines using the NACH assay.

[0087] (g,h) This figure shows the expression of (g) miRNA-21 and (h) miRNA-99a in exosomes isolated from the plasma of mice transplanted with gastric cancer cell lines through qRT-PCR analysis.

[0088] (i,j) Figure showing the fluorescence images and intensity measurements of (i) miRNA-21 and (j) miRNA-99a when total exosomal miRNA (5 μL / hydrogel) was used in the plasma of mice transplanted with gastric cancer cell lines (n = 5).

[0089] Each of the above data represents the mean ± SD of independent experiments (***p < 0.0005, **p < 0.005, *p < 0.05).

[0090] Figure 9 is a figure evaluating the performance of the NACH analysis system for the detection of miRNA extracted from tumor tissues of three mouse groups (Normal, SNU-484, and Hs 746T).

[0091] (a,b) This figure compares the expression levels of (a) miRNA-21 and (b) miRNA-99a detected using the NACH analysis system with those of the conventional qRT-PCR analysis system. The expression levels of miRNAs were normalized to the expression levels of miRNA-16 and miRNA-191 (reference genes).

[0092] (c,d) Figure showing the fluorescence images and intensity measurements corresponding to (c) miRNA-21 and (d) miRNA-99a when total miRNA (100 ng / hydrogel) isolated from mouse tumor tissue was used (n = 5).

[0093] Each of the above data represents the mean ± SD of independent experiments (***p < 0.0005).

[0094] Figure 10 is a figure confirming the performance of the NACH analysis system for clinical samples.

[0095] (a,b) This figure shows a heatmap of miRNA expression levels measured using (a) qRT-PCR and (b) NACH analysis systems in plasma samples obtained from a group of healthy individuals and a group of patients with early and advanced gastric cancer. In the heatmap, fluorescence intensity indicates the amount of miRNA-21 (green) and miRNA-99a (red) present in exosomal miRNAs.

[0096] (c,d) Figure showing the results of qRT-PCR analysis for (c) miRNA-21 and (d) miRNA-99a expression in plasma samples obtained from a group of healthy individuals and a group of patients with early and advanced gastric cancer. The expression levels of miRNAs were normalized to the levels of miRNA-16 and miRNA-191 (reference genes).

[0097] (e,f) Figure showing the results of measuring the fluorescence images and intensities of (e) miRNA-21 and (f) miRNA-99a when using total exosomal miRNA (5 μL / test) isolated from plasma samples obtained from a group of healthy individuals and a group of patients with early and advanced gastric cancer.

[0098] (g,h) This figure compares the performance of qRT-PCR and NACH analysis systems when detecting (g) miRNA-21 and (h) miRNA-99a in gastric cancer of various stages.

[0099] (i,j) Figure showing the area under the curve (AUC) values ​​for miRNA-21 and miRNA-99a in (i) early stage and (j) advanced stage gastric cancer patient groups.

[0100] Each of the above data represents the mean ± SD of independent experiments (***p < 0.0005, **p < 0.005).

[0101] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention.

[0102] Experimental Example 1. Materials

[0103] Poly(ethylene glycerol) (Mn 3,350; PEG 3.35 K), n,n-diisopropylethylamine (DIPEA), dichloromethane (DCM), and the radical photoinitiator 2-hydroxy-2-methylpropiophenone (HMPP) were obtained from Sigma-Aldrich (USA), and acryloyl chloride was sourced from Tokyo Chemical Industry Co. All oligonucleotides were purchased from Biooneer Co., CircLigase II ssDNA Ligase (100 U / μL) was purchased from Biosearch Technologies, Phi29 DNA polymerase (10 U / μL) and 10X Phi29 DNA polymerase buffer were purchased from New England Biolabs, and dNTPs (2.5 mM) and SYBR Green II were purchased from TaKaRa. Tween 20 and sodium chloride were purchased from Sigma-Aldrich, GelRed® (10,000 µm) from Biotium Co. TEMED, 10% APS, 40% acrylamide / bis solution (29:1) from Bio-Rad Co., TBE buffer (10 µm) from Biosesang, and UltraPure distilled water was purchased from Invitrogen. Additionally, RPMI-1640 medium, Dulbecco's Modified Eagle's Medium (DMEM), and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. (USA). miRNeasy® Mini Kit, exoRNeasy Midi Kit, miCRY LNA reverse transcription (RT) Kit, and miCRY LNA SYBR Green PCR Kit were purchased from Qiagen.

[0104] Experimental Example 2. Synthesis of PEG-DA

[0105] To synthesize a hydrogel backbone using PEG-DA, 60 g of PEG (molecular weight 3,350 Da) was first completely dissolved in 75 mL of DCM, and when the solution became clear, 7 mL of DIPEA was added. Subsequently, 6.5 mL of acryloyl chloride was slowly added to the mixture dropwise while stirring over ice. The mixture was stirred overnight in a dark place under vacuum at 4°C. Then, the mixture was precipitated and filtered in 1 L of diethyl ether, and the filtrate was vacuum dried to obtain a powder. The powder was dissolved in 75 mL of DCM and 500 mL of potassium carbonate (2 M), and the solution was left overnight for phase separation to remove byproducts. The lower layer was filtered, precipitated in 1 L of diethyl ether, and dried under vacuum. The synthesized PEG-DA was stored at 4°C for future use.

[0106] Experimental Example 3. Fabrication of a hydrogel containing a capture probe

[0107] First, a cylindrical hydrogel was developed through the photopolymerization of PEG-DA. A master template with a vertical column pattern was fabricated by 3D printing, and a hydrogel mold was fabricated using soft lithography with the master template and PDMS. A PDMS mixture, prepared by mixing the PDMS base and curing agent in a 10:1 weight ratio, was poured into the master template and heated at 80°C for 6 hours. 20 μL of a hydrogel precursor solution (20% [w / v] PEG-DA, 20% [w / v] PEG, 60% [v / v] 1X TE buffer, 0.1% [v / v] HMPP) was poured into the hydrogel mold, and photopolymerization was performed by irradiating with UV (254 nm) for 5 minutes. The resulting cylindrical hydrogel was rinsed with distilled water for 1 hour and stored at 4°C. After attaching acrylamide to the end of the capture probe, the capture probe was mixed with a hydrogel precursor solution, and 20 μL of the mixture was poured into a hydrogel mold to produce a hydrogel containing the capture probe. Subsequently, the unbound capture probe was removed by rinsing with distilled water for 1 hour, and the hydrogel containing the capture probe was finally obtained.

[0108] Experimental Example 4. Preparation for the Design of a NACH Assay System

[0109] NACH analysis is a system that uses rotation ring-mediated isothermal amplification, and the sequences of the DNA and probes used in the system are listed in Table 1 below.

[0110] Name Sequence (5'→3') Modification Target DNA (miRNA-21) TAG CTT ATC AGA CTG ATG TTG A (Sequence No. 1)-Target DNA (miRNA-99a) AAC CCG TAG ATC CGA TCT TGT G (Sequence No. 2)-Padlock probe (miRNA-21) GAT GTA AAT CAC AAA GAA GGG GAA TAC AAC AAC AGT CAA CAT CAG T (Sequence No. 3) 5'-Phosphate Padlock probe (miRNA-99a) GAT GTA AAT CAC AAA GAA GGG GAA TAC AAC AAC AGC ACA AGA TCG G (Sequence No. 4) 5'-Phosphate Capture probe (miRNA-21) CTG ATA AGC TAT TTT TTT TTT TTT TTT (Sequence No. 5) 3'-Acrylamide Capture probe(miRNA-99a)ATC TAC GGG TTT TTT TTT TTT TTT TTT (Sequence No. 6)3'-Acrylamide Reporter probe(miRNA-21)(FAM) GGA ATA CAA CAA CAG (Sequence No. 7)5'-FAM Reporter probe(miRNA-99a)(Cyanine 3) AGG GGA ATA CAA CAA (Sequence No. 8)5'-Cyanine 3

[0111] The total reaction volume (20 μL) consisted of circular DNA (5 μL), Phi29 DNA polymerase (1 μL), 10X Phi29 DNA polymerase buffer (2 μL), dNTPs (4 μL), reporter probe (2 μL), targets at various concentrations (in vitro, in vivo, and clinical samples), and nuclease-free water. The sample solutions were first mixed and then incubated with the previously prepared hydrogel at 37°C for 2 hours. Subsequently, unbound reporter probes were removed by washing with TET buffer containing 0.05% Tween 20 and 50 mM NaCl for 30 minutes at room temperature. Finally, Chemidoc TM Fluorescence emitted from NACH was captured and analyzed using Bio-Rad. Fluorescence intensities corresponding to miRNA-21 (λex = 495 nm, λem = 520 nm) and miRNA-99a (λex = 550 nm, λem = 570 nm) were measured, and the increase in fluorescence intensity was normalized (F / F0) and calculated.

[0112] Experimental Example 5. Cell Culture

[0113] SNU-484 (KCLB No. 00484) and Hs 746T (KCLB No. 30135) cells were purchased from the Korean Cell Line Bank (KCLB) and cultured according to the recommended methods. SNU-484 cells were cultured in RPMI-1640 medium, and Hs 746T cells were cultured in Dulbecco's modified Eagle's medium (DMEM). Both cell lines were maintained in a humidified incubator under conditions of 5% CO2 and 37°C, and 10% [v / v] FBS was supplemented in all complete culture media.

[0114] Experimental Example 6. Gastric cancer (GC) in vivo model

[0115] All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC 2019-0317) of the Yonsei University Laboratory Animal Center. Six-week-old male nude BALB / c mice (n=5, mean body weight 22±2 g) were purchased from Orient (Korea). SNU-484 and Hs 746T cells (1 × 10⁻¹⁰) resuspended in 60 μL of serum-free medium 7 Cells were independently injected into the proximal thigh region of male nude BALB / c mice using a 29G insulin syringe.

[0116] Tumor volume 1000 mm 3 Mice were observed until [value] was reached or ulcers developed. Tumor size was evaluated 3 times per week, and [(4 / 3)ХпХ(secondary axis / 2) 2 Х(Main axis / 2) mm 3 It was calculated using the ] formula. Untreated healthy male nude BALB / c mice (n = 5) were used as a control. Plasma and tissue samples were collected from the mice for subsequent analysis.

[0117] Experimental Example 7. miRNA Extraction

[0118] Cellular miRNA was extracted using the miRNeasy kit (Qiagen) according to the manufacturer's instructions. Specifically, mouse tumors were lysed in QIAzol lysis reagent using a tissue lysator, and total RNA was extracted using the miRNeasy kit (Qiagen). Exosomal miRNA isolated from 100 μL of human and mouse plasma was extracted and purified using the exoRNeasy Midi kit (Qiagen). Subsequently, the extracted RNA was treated with Nanodrop One TM It was quantified using Thermo Fisher Scientific.

[0119] Experimental Example 8. Real-time Polymerase Chain Reaction (qRT-PCR)

[0120] The previously purified miRNA was reverse transcribed into cDNA using the miRCURY® LNA® RT kit (Qiagen). The RT reaction (10 μL) consisted of 5-X miRCURY RT reaction buffer (2 μL), 10-X miRCURY RT enzyme mixture (2 μL), miRNA (1 μL), and nuclease-free water (5 μL). The RT cycle conditions involved inactivation at 42°C for 60 minutes and 95°C for 5 minutes. The synthesized cDNA was stored at 4°C for future use. The qRT-PCR system consisted of 2-X miRCURY SYBR Green master mix (5 μL), PCR primer mix (1 μL), ROX reference dye (0.05 μL), diluted cDNA template (2 μL), and nuclease-free water (1.95 μL). The reaction was performed using CFX96 TM It was performed on a real-time PCR system (Bio-Rad). The following cycling conditions were used: 2 minutes at 95°C, followed by 45 cycles of 10 seconds at 95°C and 60 seconds at 56°C. The expression of the target miRNA was normalized to the expression of miRNA-16 and miRNA-191.

[0121] Experimental Example 9. Polyacrylamide Gel Electrophoresis (PAGE)

[0122] PAGE samples were run on a 10% natural polyacrylamide gel under conditions of TBE buffer, 60V voltage, and 120 minutes. Subsequently, the gel was stained with GelRed for 30 minutes, and the images were taken with Chemidoc. TM It was captured via (Bio-Rad).

[0123] Experimental Example 10. Clinical Sample Collection

[0124] Plasma samples (n = 50) collected from GC patients were provided by the Ajou University Hospital Biobank (Approval No. AJHB-2023-03), and the study protocol was approved by the Ethics Committee of the Korea Research Institute of Bioscience and Biotechnology (IRB #2022-1317-077). The study included 40 patients clinically diagnosed with cancer and 10 healthy individuals, and patient samples were classified into early and advanced stages based on pathological results obtained after endoscopic mucosal resection.

[0125] Experimental Example 11. Statistical Analysis

[0126] All experiments were performed independently at least three times, and the number of repetitions was indicated on the graph of each figure. Data were reported as mean ± SD, and the following legend was added to each figure: ***p < 0.0005, **p < 0.005, *p < 0.05. LOD was calculated according to the following equation: LOD = 3 σ / m, where σ is the SD of the blank sample and m represents the slope of the data fitting the linear range.

[0127] Example 1. Design of NACH Analysis System

[0128] The nucleic acid amplification circuit-based hydrogel assay (NACH assay) system is an enzymatic cascade reaction system utilizing rolling circle amplification (RCA) reactions, designed to stably detect target miRNAs through exponential amplification reactions. In the present invention, NACH assays were performed targeting miRNA-21, a tumor-associated gene, and miRNA-99a, which promotes EMT (Fig. 1(a)).

[0129] Specifically, the NACH analysis system of the present invention hybridized circular DNA, target miRNA, and a capture probe into a hydrogel for one-step detection. To facilitate the amplification reaction through the hybridization of the probes within the hydrogel, an acrylamide group was attached to the 3' end of the capture probe and then made to interact with polyethylene glycol diacrylate (PEG-DA), a hydrogel precursor. Half of the target miRNA bound to the capture probe within the hydrogel, and the other half bound to the circular DNA to initiate the amplification reaction within the hydrogel. The amplification product generated by RCA was bound to a reporter probe. The reporter probe used for miRNA-21 detection was 5'-labeled with Fluorescein amidite (FAM), and the reporter probe used for miRNA-99a detection was 5'-labeled with Cyanine3 (CY3). Each reporter probe showed a fluorescent signal when it bound to the amplification products of miRNA-21 and miRNA-99a in the RCA reaction (Fig. 1(b)).

[0130] Example 2. Optimization of NACH Analysis System

[0131] Circular DNA was formed through the circularization of the padlock probe via phosphodiester bonding, which was confirmed by PAGE (Fig. 2(a)).

[0132] To optimize the enzyme concentrations critical for the RCA reaction, experiments were conducted with dNTP concentrations ranging from 100 to 500 μM and Phi29 DNA polymerase concentrations ranging from 2 to 12 units, and the results were compared (Fig. 3). Based on this, the optimal conditions were determined to be a dNTP concentration of 400 μM and a Phi29 DNA polymerase concentration of 10 units. Meanwhile, it was confirmed that fluorescence intensity has a positive correlation with the concentration of the target miRNA (Figs. 2(b) and 2(d)).

[0133] The amplification products generated by the RCA reaction were analyzed via native PAGE (Figs. 2(c) and 2(e)), and the hybridization and amplification products between the circular DNA, target miRNA, and capture probe were analyzed via PAGE in the NACH analysis system (Fig. 4). In the process for the synthesis of the capture probe and PEG-DA-based hydrogel (Fig. 2(f)), the PEG-DA was 1732 cm⁻¹ -1 A strong absorption peak corresponding to the carbonyl group (C=O) of the acrylate was observed (Fig. 5).

[0134] Reporter probe concentration conditions for the detection of miRNA-21 and miRNA-99a were optimized using reporter probes at various concentrations (0 to 1000 nM) (Figs. 2(g) and 2(h)). Based on these experiments, miRNA-21 and miRNA-99a were detected using reporter probes at concentrations of 1000 nM for miRNA-21 and 500 nM for miRNA-99a, respectively.

[0135] In summary, it was confirmed that the most suitable concentrations for the NACH analysis system were 400 μM for dNTPs, 10 units for Phi29 DNA polymerase, 1000 nM for miRNA-21, and 500 nM for miRNA-99a.

[0136] Example 3. Sensitivity and Specificity of the NACH Analysis System

[0137] The sensitivity of the NACH assay system was evaluated using target miRNAs at various concentrations (1 fM to 100 nM), and ChemiDoc TMThe fluorescence intensity of NACH was analyzed using the system. As a result, the fluorescence intensity was linearly proportional to the logarithm of the target miRNA concentration (Figs. 6(c) and 6(d)). Based on this, the limit of detection (LOD) was calculated, and the LOD for miRNA-21 and miRNA-99a was 1 fM, confirming that it is similar to previously studied isothermal amplification systems in terms of detection time and sensitivity.

[0138] In addition, the specificity of the NACH analysis system was evaluated using genes with 1 bp and 2 bp mismatched sequences and cross-validation sequences as shown in Table 2 below as negative controls. As a result, high fluorescence intensity was observed for the target miRNA, confirming that the detection specificity of the NACH analysis system was high (Figs. 6(e) and 6(f)).

[0139] NameSequence (5'→3')1 bp mismatch(miRNA-21)TAG CTT ATC AGA CTG ATGCTG A (Sequence No. 9)2 bp mismatch(miRNA-21)TAG CTT ATC AGA CTG ATTCTG A (Sequence No. 10)1 bp mismatch(miRNA-99a)AAC CCATAG ATC CGA TCT TGT G (Sequence No. 11)2 bp mismatch(miRNA-99a)AAC CCATGG ATC CGA TCT TGT G (Sequence No. 12)Cross-validation sequence(miRNA-21)AAC CCG TAG ATC CGA TCT TGT G (Sequence No. 13)Cross-validation sequence(miRNA-99a)TAG CTT ATC AGA CTG ATG TTG A (Sequence No. 14)

[0140] Sequences that do not match the target miRNA sequence are indicated in bold.

[0141] In other words, the NACH analysis system according to the present invention was found to have performance similar to existing isothermal amplification systems in terms of detection time and sensitivity, while exhibiting high detection specificity, confirming that it is a system with excellent performance for target nucleic acid detection.

[0142] Example 4. Performance of the NACH analysis system on preclinical samples

[0143] The performance of the NACH analysis system was evaluated using gastric cancer cell lines SNU-484 and Hs 746T as preclinical samples.

[0144] Specifically, the expression of miRNA-21 and miRNA-99a in SNU-484 and Hs 746T cell lines was analyzed using a NACH assay system. As a result, while weak fluorescence was observed in the non-target control (NTC; RNA-free buffer), strong fluorescence was observed specifically in miRNA-21 in Hs 746T cells and specifically in miRNA-99a in SNU-484 cells (Fig. 7).

[0145] As a result of observing the expression of exosomal miRNAs extracted from each cell culture medium, it was confirmed that an expression pattern similar to that observed in intracellular RNA appeared. An in vivo model was prepared by transplanting SNU-484 and Hs 746T cell lines into mice, and exosomal RNA extracted from plasma isolated from said model was applied to a qRT-PCR and NACH analysis system, and it was confirmed that a similar expression pattern appeared (Fig. 8).

[0146] In addition, RNA extracted by homogenizing tumor tissue excised from the above-prepared in vivo model was applied to qRT-PCR and NACH analysis systems. In qRT-PCR, miRNA-21 was overexpressed 25.0-fold in the tumor tissue of the Hs746T-transplanted model, and miRNA-99a was overexpressed approximately 1.6-fold in the tumor tissue of the SNU-484-transplanted model (Figs. 9(a) and 9(b)), and each target miRNA was also detected with high fluorescence intensity in the NACH analysis system (Figs. 9(c) and 9(d)).

[0147] That is, through experiments using SNU-484 and Hs 746T as preclinical samples, it was confirmed that the NACH analysis system according to the present invention has excellent performance in detecting miRNA-21 and miRNA-99a.

[0148] Example 5. Performance of the NACH analysis system on clinical samples

[0149] Blood (plasma) samples were obtained from early and advanced stage gastric cancer patients (n = 20 each) and healthy individuals (n = 10), and exosomal miRNA (5 μL) extracted from the samples was applied to a NACH analysis system. As a result, it was confirmed that miRNA-21 and miRNA-99a were upregulated in the gastric cancer patient groups (Early, Progressive) compared to the healthy individual groups (Healthy, Normal) (Fig. 10).

[0150] Therefore, it was confirmed that the NACH analysis system of the present invention is a more useful platform for gastric cancer diagnosis than qRT-PCR.

Claims

1. A composition for detecting a target nucleic acid, comprising a hydrogel comprising: (i) a capture probe having a sequence complementary to a portion of the target nucleic acid and having its ends immobilized on the hydrogel; (ii) circular DNA having a sequence complementary to a portion of the target nucleic acid; and (iii) a reporter probe that binds to an amplification product.

2. A composition for detecting a target nucleic acid according to claim 1, further comprising dNTPs and nucleic acid polymerase for an RCA reaction.

3. A composition for detecting a target nucleic acid according to claim 2, wherein the nucleic acid polymerase is any one selected from the group consisting of Phi29 DNA polymerase, phage M2 DNA polymerase, phage Phi-PRD1 DNA polymerase, VENT.RTM DNA polymerase, Klenow fragment DNA polymerase I, T5 DNA polymerase, PRD1 DNA polymerase, T4 DNA polymerase holoenzyme, T7 native polymerase, and Bst polymerase.

4. A composition for detecting target nucleic acids according to claim 1, wherein the capture probe has one or more groups selected from the group consisting of acrylamide, glutaraldehydes, and adipic acid dihydrazide attached to its 3' end.

5. A composition for detecting a target nucleic acid according to claim 1, wherein the reporter probe comprises any one selected from the group consisting of ALEX-350, FAM, VIC, TET, CAL Fluor®Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY5, CY5.5, Quasar 705, Fluorescein amidite (FAM), and Cyanine3 (Cy3).

6. A composition for detecting a target nucleic acid according to claim 1, wherein half of the target nucleic acid binds to a capture probe and the other half binds to circular DNA.

7. (a) A step of treating a sample on a hydrogel comprising: (i) a capture probe having a sequence complementary to a portion of the target nucleic acid and having its ends immobilized on the hydrogel; (ii) circular DNA having a sequence complementary to a portion of the target nucleic acid; and (iii) a reporter probe that binds to the amplification product; (b) a step in which the target nucleic acid of the above sample is amplified; and (c) a step of binding a reporter probe to the target nucleic acid amplified in step (b) to generate a detectable signal; a method for detecting a target nucleic acid, comprising:

8. A method for detecting a target nucleic acid according to claim 7, wherein the detectable signal is a fluorescent signal emitted by a reporter probe.

9. A method for detecting a target nucleic acid according to claim 7, wherein the target nucleic acid is any one selected from the group consisting of miRNA, miRNA sponge, tough decoy miRNA, anti-miR, small RNA, siRNA, and shRNA.

10. A method for detecting a target nucleic acid according to claim 7, wherein the target nucleic acid amplification in step (b) above is amplified by a rolling circle amplification (RCA) reaction of the target nucleic acid of the sample.

11. A method for detecting a target nucleic acid according to claim 10, comprising dNTPs and a nucleic acid polymerase for an RCA reaction.

12. A method for detecting a target nucleic acid according to claim 7, wherein the capture probe has one or more groups selected from the group consisting of acrylamide, glutaraldehydes, and adipic acid dihydrazide attached to its 3' end.

13. A method for detecting a target nucleic acid according to claim 7, wherein the reporter probe comprises any one selected from the group consisting of ALEX-350, FAM, VIC, TET, CAL Fluor®Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY5, CY5.5, Quasar 705, Fluorescein amidite (FAM), and Cyanine3 (Cy3).

14. (a) A step of treating a sample on a hydrogel comprising: (i) a capture probe having a sequence complementary to a portion of the target nucleic acid and having its ends immobilized on the hydrogel; (ii) circular DNA having a sequence complementary to a portion of the target nucleic acid; and (iii) a reporter probe that binds to the amplification product; (b) a step in which the target nucleic acid of the above sample is amplified; and (c) a step in which a reporter probe binds to the target nucleic acid amplified in step (b) to generate a detectable signal; comprising a method for providing information about cancer.

15. A method for providing information about cancer, wherein the detectable signal in paragraph 14 is a fluorescent signal emitted by a reporter probe.

16. A method for providing information on cancer according to claim 14, wherein the cancer is any one selected from the group consisting of stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, and gallbladder cancer.

17. A method of providing information about cancer, wherein the cancer in paragraph 16 is stomach cancer.

18. A method for providing information on cancer, wherein, in paragraph 14, the target nucleic acid amplification of step (b) above is amplified by a rolling circle amplification (RCA) reaction in which the target nucleic acid of the sample is amplified.

19. A method for providing information about cancer according to paragraph 18, comprising dNTPs and nucleic acid polymerase for an RCA reaction.

20. In paragraph 14, the capture probe has one or more groups selected from the group consisting of acrylamide, glutaraldehydes, and adipic acid dihydrazide attached to its 3' end, and A method for providing information about cancer, wherein the reporter probe comprises any one selected from the group consisting of ALEX-350, FAM, VIC, TET, CAL Fluor®Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY5, CY5.5, Quasar 705, Fluorescein amidite (FAM), and Cyanine3 (Cy3).

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