[go: up one dir, main page]

WO2024096642A1 - Aptamère adn pour le diagnostic ou le traitement du cancer colorectal - Google Patents

Aptamère adn pour le diagnostic ou le traitement du cancer colorectal Download PDF

Info

Publication number
WO2024096642A1
WO2024096642A1 PCT/KR2023/017433 KR2023017433W WO2024096642A1 WO 2024096642 A1 WO2024096642 A1 WO 2024096642A1 KR 2023017433 W KR2023017433 W KR 2023017433W WO 2024096642 A1 WO2024096642 A1 WO 2024096642A1
Authority
WO
WIPO (PCT)
Prior art keywords
aptamer
sev
sevs
dna aptamer
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2023/017433
Other languages
English (en)
Korean (ko)
Inventor
박기수
차병석
이은성
장영준
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University Industry Cooperation Corporation of Konkuk University
Original Assignee
University Industry Cooperation Corporation of Konkuk University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020220144790A external-priority patent/KR20240066514A/ko
Application filed by University Industry Cooperation Corporation of Konkuk University filed Critical University Industry Cooperation Corporation of Konkuk University
Priority claimed from KR1020230150082A external-priority patent/KR20250064751A/ko
Publication of WO2024096642A1 publication Critical patent/WO2024096642A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention is a technology related to a DNA aptamer for colon cancer diagnosis and treatment.
  • colon cancer can be diagnosed with high affinity and specificity, and it specifically binds to colon cancer-derived sEV to prevent sEV-mediated cancer metastasis. It is a technology that has been confirmed to have therapeutic potential as it has the potential to suppress it.
  • the present invention also provides a technology related to a DNA aptamer that can diagnose colon cancer with high heterogeneity characteristics, and a technology that confirms the possibility of colon cancer diagnosis with high affinity and specificity using an aptamer.
  • This invention is as part of the individual basic research of the Ministry of Science and ICT (Ministry of Science and ICT) [Project identification number: 1711157003, task number: 2020R1C1C1012275, research project name: Exosome SELEX technology (E-SELEX) and cancer diagnosis bio using the same] It is derived from research conducted.
  • Colorectal cancer a threat to global health care, is the third most diagnosed cancer and the second most common cause of cancer death worldwide. In 2020, colorectal cancer accounted for 10% of cancer diagnoses and 9.4% of cancer deaths worldwide. The neoplastic progression of colorectal cancer is initiated by a combination of factors, including genetic mutations, epigenetic modifications, and environmental factors, ultimately promoting recurrence and metastasis in various organs, especially the liver. Colon cancer is known to be a carcinoma that is particularly difficult to diagnose and treat because it is characterized by high tumor heterogeneity.
  • small extracellular vesicles also known as exosomes
  • sEVs are nano-sized lipid membrane-enclosed endoplasmic reticulum (50-200 nm in diameter) that are secreted extracellularly by all cell types.
  • sEVs are stable in various body fluids such as blood, urine, sweat, and saliva.
  • sEVs transport active biomolecules such as DNA, RNA, and proteins to locally and systematically mediate intercellular signaling between cells of origin and recipient cells.
  • sEVs derived from tumor cells can act as regulators of cancer development, progression, invasion, and metastasis. More and more studies have demonstrated the potential applications of CRC-derived sEVs.
  • sEVs microRNAs
  • lncRNAs long non-coding RNAs
  • mRNAs messenger RNAs
  • candidate sEVs biomarkers for CRC prognosis including metastasis, chemoresistance, and recurrence, were identified. Since sEVs circulating in human body fluids are considered mini versions of parent cells, non-invasive diagnosis of colorectal cancer is possible. Therefore, there is increasing research interest in using sEVs as a promising tool for diagnostic and therapeutic applications. In particular, it is very important to distinguish between colon cancer-derived sEVs and normal human serum-derived sEVs, whose origin is difficult to accurately determine.
  • aptamers which are single-stranded DNA or RNA that can bind to a specific target, are widely used in diagnostic and therapeutic development and biotechnology due to their outstanding advantages such as high stability, small size, ease of synthesis and modification, low immunogenicity, and high binding affinity and specificity. It is receiving great attention when it comes to marker discovery.
  • Aptamers are typically screened through an iterative in vitro selection process called Systematic Evolution of Ligands by EXponential Enrichment (SELEX).
  • SELEX Systematic Evolution of Ligands by EXponential Enrichment
  • the present inventors found a stable DNA aptamer that can diagnose colon cancer through sEVs, revealed its potential to treat colon cancer by inhibiting sEVs-mediated cancer metastasis, and demonstrated various properties that can be used to diagnose colon cancer.
  • the present invention was completed by finding a DNA aptamer with high binding to colon cancer sEVs and using it to develop a highly sensitive biosensor.
  • the purpose of the present invention is to provide at least one DNA aptamer for colon cancer diagnosis selected from the group consisting of base sequences of SEQ ID NOs: 4 to 17.
  • Another object of the present invention is to provide a composition for diagnosing colon cancer, a kit, and a colon cancer-specific drug delivery composition containing the aptamer.
  • Another object of the present invention is to provide a method for producing the aptamer, a method for providing information for colon cancer diagnosis using the aptamer, and a method for detecting surface biomarkers specific to colon cancer sEVs.
  • Another object of the present invention is to provide at least one DNA aptamer for colon cancer diagnosis selected from the group consisting of base sequences of SEQ ID NOs: 18 to 35.
  • Another object of the present invention is to provide a composition for diagnosing colon cancer, a kit, and a colon cancer-specific drug delivery composition containing the aptamer.
  • Another object of the present invention is to provide a method for producing the aptamer, a method for providing information for colon cancer diagnosis using the aptamer, and a method for detecting surface biomarkers specific to colon cancer sEVs.
  • the present inventors used colon cancer sEV SELEX; E-SELEX was built. After proceeding with a total of three E-SELEX loops, where each loop consists of four consecutive positive selections and one counter selection, the top 10 aptamer candidates are selected and subjected to enzyme-linked oligonucleotide assay (ELONA). ) method was used to evaluate binding affinity. Finally, the best aptamer was obtained through post-SELEX optimization, which resulted in a K d value of 3.41 nM and a detection limit of 1.0 It was confirmed that it had sufficient sensitivity for detection.
  • E-SELEX enzyme-linked oligonucleotide assay
  • sEVs derived from healthy serum and sEVs from normal cells, and sEVs derived from other cancer cells
  • the selected optimal aptamers were further investigated to predict biomarkers of sEVs derived from colon cancer cells, and were demonstrated to inhibit cell migration and proliferation by regulating sEV-mediated intercellular signaling.
  • TEV-SELEX Colorectal Cancer
  • SW620, LS 174T, HT29 colon cancer cells
  • TEV-SELEX Colorectal Cancer
  • SW620, LS 174T, HT29 three types of colon cancer cells
  • HT29 colon cancer cells
  • TEV-SELEX was constructed with a total of eight loops, with each loop consisting of three consecutive positive selections and one counter selection. Based on the NGS results, the top 10 aptamer candidates by frequency (%) were selected and enzyme-linked. Binding affinity was evaluated using an oligonucleotide assay (Enzyme-linked oligonucleotide assay, ELONA) method.
  • ELONA Endome-linked oligonucleotide assay
  • the present invention provides a DNA aptamer for diagnosing or treating colon cancer selected from the group consisting of the base sequences of SEQ ID NOs: 4 to 17, and one or more colon cancers selected from the group consisting of the base sequences of SEQ ID NOs: 18 to 35 Provides DNA aptamers for cancer diagnosis.
  • the present invention relates to the treatment or diagnosis of colon cancer of a DNA aptamer containing at least one sequence selected from the group consisting of the base sequences of SEQ ID NOs: 4 to 17, and the use of a DNA aptamer selected from the group consisting of the base sequences of SEQ ID NOs: 18 to 35.
  • a DNA aptamer containing at least one sequence is provided for use in colon cancer diagnosis.
  • colon cancer is used with the same meaning as “colorectal cancer,” “Colorectal cancer,” and “CRC.”
  • DNA aptamer refers to a single-stranded oligonucleotide that has the characteristic of binding to a target with high affinity and specificity and each has a unique three-dimensional structure. Through repeated in vitro selection and enrichment processes, it is possible to select DNA molecules that specifically bind to a specific target, that is, DNA aptamers, from a DNA aptamer library.
  • Diagnosis means confirming the presence or characteristics of a pathological condition. Diagnosis in the present invention is to confirm the presence or occurrence of colon cancer using a DNA aptamer.
  • treatment means any beneficial action, such as improving the symptoms or suppressing the progression of colon cancer that has already been caused by using the DNA aptamer of the present invention.
  • it may include a DNA aptamer having 90% or more sequence homology to the nucleotide sequences of SEQ ID NOs. 4 to 35, and the term “base sequence having 90% or more sequence homology” ranges from 1 to 10. It refers to a nucleotide sequence that shows similar cancer-specific binding ability by adding, deleting, or substituting 90% or more but less than 100% of the sequences with nucleotides added, deleted, or substituted.
  • the aptamer can specifically bind to sEVs derived from colon cancer cells.
  • sEVs were isolated from positive and counter targets, and when the DNA aptamer of the present invention was used, it did not have binding specificity to sEVs derived from normal colon cells, human breast cancer cells, or human glioblastoma. , it was confirmed that it specifically bound only to sEV derived from colon cancer cells (Figure 8).
  • the aptamer of the present invention may further include one or more selected from the group consisting of forward primer and reverse primer sequences.
  • a "primer” is a nucleic acid sequence with a short free 3' terminal hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template. It refers to a short nucleic acid sequence that performs a function.
  • colon cancer can be diagnosed by performing PCR amplification using the forward and reverse primers of the DNA aptamer described above. PCR conditions and lengths of forward and reverse primers can be modified based on those known in the art.
  • the sequence of the forward primer may consist of the base sequence of SEQ ID NO: 2
  • the sequence of the reverse primer may consist of the base sequence of SEQ ID NO: 3, but are not limited thereto.
  • the primer region was used for PCR amplification by including a forward primer in the 5' portion of the aptamer sequence and a reverse primer region in the 3' portion of the aptamer sequence in an ssDNA library during the E-SELEX or TEV-SELEX process. It is obvious to those skilled in the art that primer regions can be combined at the 5' and 3' portions for PCR amplification.
  • the DNA aptamer may consist of the base sequence of SEQ ID NO: 13.
  • the top 10 candidate aptamers by frequency (%) after SELEX were selected, and aptamers with binding affinity and specificity were selected.
  • CCE-10 had the highest affinity for sEV.
  • the DNA aptamer consisting of the base sequence of SEQ ID NO: 13 may be additionally linked to one or more of the forward primer base sequence and the reverse primer base sequence.
  • the DNA aptamer consisting of the base sequence of sequence number 15 with the forward primer removed showed the highest affinity. Therefore, the DNA aptamer may be composed of the base sequence of sequence number 15.
  • the binding dissociation constant may be 3 to 4 nM, preferably 3.2 to 3.6.
  • the DNA aptamer may consist of the base sequence of SEQ ID NO: 32.
  • the top 10 candidate aptamers by frequency (%) after SELEX were selected, and aptamers with high binding affinity and specificity were selected.
  • T6 had the highest affinity for sEVs.
  • optimization of the T6 aptamer sequence was performed. Therefore, as shown in [Table 8], the T6F T6R T6FR T6RA T6RB T6RB1 T6RB2 T6RB3 sequence was used.
  • the present invention also provides a composition for diagnosing or treating colon cancer containing the aptamer above.
  • the composition may further include physiologically acceptable excipients, carriers, or additives, including starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate. , white sugar, dextrose, sorbitol, and talc may be used, but are not limited thereto.
  • physiologically acceptable excipients, carriers, or additives including starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn star
  • composition containing the DNA aptamer when using a composition containing the DNA aptamer, it hardly binds to normal serum, normal colon cell lines, or other human cancer cell lines other than colon cancer cell lines, so it can be used to specifically target (various) colon cancer cell lines. Detection was confirmed.
  • the present invention also provides a kit for diagnosing or treating colon cancer, comprising the composition.
  • a colon cancer diagnosis kit containing an aptamer that specifically binds to colon cancer-derived sEV.
  • This colon cancer diagnostic kit may include a buffer solution and containers for detection and analysis as needed, such as bottles, tubs, sachets, envelopes, tubes, and ampoules. They may be formed in part or entirely from plastic, glass, paper, foil, wax, etc.
  • the container may be equipped with a completely or partially removable closure that may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means.
  • the container may also be equipped with a stopper, allowing access to the contents by means of a needle.
  • the kit may include an external package, and the external package may include instructions for use of the components.
  • the kit may be a diagnostic sensor, RT-PCR kit, competitive RT-PCR kit, real-time RT-PCR kit, DNA chip kit, and protein chip kit.
  • the kit of the present invention may include a composition, solution, or device containing not only primers and probes that recognize the DNA aptamer, but also one or more other components suitable for the analysis method.
  • the DNA aptamer that specifically binds to the colon cancer-derived sEV may be used on conventional supports such as beads, particles, dipsticks, fibers, filters, membranes, and glass slides, and solid supports such as silane or silicate supports. By being fixed to and provided as a detection sensor, it can be used for colon cancer diagnosis.
  • the present invention may be a sensor for colon cancer diagnosis in which a DNA aptamer that specifically binds to the colon cancer-derived sEV is fixed.
  • the solid support includes at least one substantially rigid surface, on which the DNA aptamers can be immovably fixed.
  • the DNA aptamer can be immobilized by any conventional chemical coupling method.
  • biotin is bound to the end of the DNA aptamer to form a complex
  • streptavidin or avidin is immobilized on the surface of a substrate such as the chip, so that the interaction between the biotin and streptavidin or avidin immobilized on the substrate surface
  • the DNA aptamer can be immobilized on the surface of the substrate.
  • kits can be applied to the kit as a signal generation method to detect DNA aptamers bound to colon cancer sEVs. Examples include colorimetry, fluorescence, and electrochemistry, but are not limited thereto, and any method that can be implemented through a labeling method suitable for each signal generation can be used without limitation.
  • the DNA aptamer can be administered to the subject in various forms according to the selected administration route as understood by those skilled in the art through the above-described itself, composition, or kit. there is.
  • it may be administered by topical, enteral or parenteral application.
  • Topical applications include, but are not limited to, epidermis, inhalation, enemas, eye drops, ear drops, and application through mucous membranes within the body.
  • Enteral applications may include oral administration, rectal administration, vaginal administration, and gastric feeding tubes.
  • Parenteral administration is intravenous, intraarterial, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, intrasternal, intraperitoneal, subcutaneous, Methods of administration may include intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical.
  • the DNA aptamer itself, the composition, or the kit described above may be formulated in an appropriate form depending on the route of administration, etc. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.
  • the present invention also provides a method for producing the DNA aptamer.
  • the method is
  • the aptamer in the method of producing a DNA aptamer, can specifically bind to sEV derived from colon cancer cells, but this description is omitted to avoid duplication as described above in the description of the DNA aptamer.
  • the present invention also provides a method of providing information for diagnosis or treatment of colon cancer using the DNA aptamer.
  • the present invention provides a method for detecting colorectal cancer sEV-specific surface biomarkers using the aptamer.
  • the method may include contacting the DNA aptamer with samples such as colon tissue, colon cells, blood, serum, plasma, saliva, sputum, and urine.
  • samples such as colon tissue, colon cells, blood, serum, plasma, saliva, sputum, and urine.
  • the sample is isolated from a mammal, preferably from the human body, and may be a sample that can be obtained with minimal invasiveness, secreted body fluid, or in vitro cell culture component sample, but preferably may be sEV derived from colon cancer cells, and may be a colon cancer marker. It is obvious that the sample is not limited to the above as long as it is a sample that may be included.
  • Colon cancer biomarkers can be separated through analysis of substances bound to the DNA aptamer in the sample, and the present invention can be used to detect colon cancer cell-specific surface biomarkers using the DNA aptamer. .
  • the present invention also provides a colon cancer-specific drug delivery composition containing the aptamer.
  • aptamers can be used to treat colon cancer by specifically binding to sEVs derived from colon cancer cells and inhibiting the progression of cancer through sEVs. Therefore, the present invention Since the aptamer will specifically bind to sEV derived from colon cancer cells and inhibit the mechanism of colon cancer progression, it is known to those skilled in the art that a composition containing this can be provided as a composition for treating colon cancer. It is self-explanatory.
  • aptamers to the surface of liposomes or nanoparticles, anticancer drugs, toxins, cancer growth inhibitory genes, siRNA (small interfering RNA), etc. loaded inside liposomes or nanoparticles can be selectively delivered to sEVs derived from colon cancer cells.
  • the aptamer according to the present invention includes known colon cancer-specific drugs, toxins and anticancer agents that induce cancer cell death, or Herpes simplex virusthymidine kinase (HSV-TK), cytosine deaminase (CD), etc.
  • HSV-TK Herpes simplex virusthymidine kinase
  • CD cytosine deaminase
  • siRNA small interfering RNA
  • the present invention provides the DNA aptamer It may be provided in the form of a colon cancer-specific drug delivery composition containing.
  • an aptamer that specifically binds only to colon cancer cell-derived cells can be used for diagnosis and treatment of colon cancer.
  • Figure 1 shows characterization of isolated sEVs.
  • Figure 1 (A) shows the NTA results for SW620 sEV (insert: cryo-TEM image), and Figure 1 (B) shows the NTA results for HS (human serum) sEV. Scale bar of cryo-TEM image: 100 nm.
  • Figure 1 (C) shows the results of Western blot analysis of CD63, CD9, Hsp90 ⁇ , and calnexin expression.
  • Figure 2 shows the workflow of (A) E-SELEX and (B) E-SELEX, respectively.
  • Figure 3 shows an agarose (2.5%) gel image for comparing symmetric and asymmetric PCR results.
  • M 25/100 bp DNA size marker; 1 and 2: w/o and w/ssDNA templates in symmetric PCR; Lanes 3 & 4: w/o and w/ssDNA templates from asymmetric PCR; Lane 5: ssDNA template (comparison)
  • Figure 4 shows the E-SELEX monitoring results using qPCR.
  • A Amplification curve.
  • B Melting curve analysis.
  • Figure 5 shows the ratio of F20th 20th cycle fluorescence intensity to Fmax maximum fluorescence intensity ( F20th / Fmax ) to estimate the degree of homogeneity.
  • Figure 6 shows the results of aptamer evaluation using the ELONA method.
  • Figure 6 (A) shows the binding affinity of the top 10 candidate aptamers
  • Figure 6 (B) shows the binding affinity of CCE-10 with and without the primer region.
  • the concentration of aptamer used in the binding affinity analysis was 250 nM.
  • Figure 7 shows the dissociation constant ( K d ) curves of CCE-10F (A) and CCE-10FR (B).
  • Figure 8 shows the evaluation results of the optimized aptamer CCE-10F.
  • Figure 8 (A) Two-dimensional and three-dimensional shapes of the predicted secondary structure of the aptamer,
  • Figure 8 (B) Binding affinity comparison with that in any ssDNA library,
  • Figure 8 (D) This is the specificity evaluation result, and the concentration of the aptamer used in the evaluation was 10 nM.
  • Figure 8 (E) shows the stability analysis results of aptamer (Apt) and sEV-bound aptamer (Apt-Exosomes) in human serum, showing the band intensity (left) and each stability (right) of the aptamer at each time point. This is a representative gel image shown.
  • Figure 9 shows the results of biomarker topology analysis. I: intact sEV, II: proteinase K (proK) treated, III: trypsin treated sEV, IV: RIPA treated sEV.
  • Figure 10 shows the results of cytotoxicity analysis. This is the analysis result for LS 174T in Figure 10 (A) and HUVEC in Figure 10 (B).
  • Figure 11 shows the results of sEV absorption analysis by LS 174T.
  • Figure 11 shows (A) confocal microscopy image, (B) flow cytometry result; Representative histograms (left) and fluorescence quantification (right) are shown, scale bar of microscopy images: 10 ⁇ m.
  • Figure 12 shows the results confirming the effect of sEV and Apt-sEV on the migration and invasion of LS 174T.
  • A representative images of wound healing assay at 0 h and 24 h (scale bar: 200 ⁇ m)
  • B quantitative analysis of gap closure
  • C representative image of transwell migration assay at 48 h (scale bar: 100 ⁇ m)
  • D quantitative analysis of migration area
  • E representative image of transwell invasion analysis at 48 hours (scale bar: 100 ⁇ m)
  • F quantitative analysis of invasion area.
  • Figure 13 shows the results confirming the effect of sEV and Apt-sEV on HUVEC angiogenesis.
  • Figure 13 shows (A) representative images of the tube formation assay at 4 h (scale bar: 100 ⁇ m), (B-C) quantitative analysis results of the relative number of junctions (B) and relative total branch length (C), respectively.
  • Figure 14 shows characterization of isolated sEVs.
  • Figure 14 (A) shows NTA results for SW620 sEV (insert: cryo-TEM image)
  • Figure 14 (B) shows NTA results for LS 174T sEV (insert: cryo-TEM image)
  • Figure 14 (C) shows the NTA results for HT29 sEVs (insert: cryo-TEM image)
  • Figure 14 (E) shows the results of Western blot analysis for the expression of CD63, Hsp90 ⁇ , and calnexin, and the scale bar of each cryo-TEM image is 100 nm.
  • Figure 15 shows the workflow of Colorectal Cancer Toggle sEV-SELEX (CRC TEV-SELEX) performed according to an embodiment of the present invention.
  • Figure 16 shows TEV-SELEX monitoring results.
  • Figure 16 (A) is an amplification curve
  • Figure 3 (B) is the ratio of F20th 20th cycle fluorescence intensity to Fmax maximum fluorescence intensity for estimating the degree of homogeneity of ssDNA from random library (Lib) and 1st to 8th loops. (F 20th /F max )
  • Figure 17 shows the binding affinity analysis results of the top 10 candidate aptamers (T1 to T10) selected according to the present invention.
  • Figure 18 shows the results of binding affinity analysis of aptamer candidates according to the present invention.
  • Figure 18 (A) shows the binding affinity of T6 and its derivative sequences (T6F, T6R, and T6FR), and
  • Figure 18 (B) to (D) shows the binding affinity of the positive target (SW620, LS 174T, The binding affinity is shown along with the ratio of T6 and its derived sequences to HT29).
  • Figure 19 shows the structural schematic diagram and binding affinity of the aptamer and its derived sequences according to the present invention.
  • Figures 19 (A) and (B) show the structure and binding affinity of T6R and its truncated versions of derived sequences (T6RA, T6RB), and
  • Figures 19 (C) and (D) show T6RB and its truncated versions.
  • the structure and binding affinity of the derived sequences (T6RB1, T6RB2, T6RB3) are shown.
  • Figure 20 shows the evaluation results of T6RB, the optimal aptamer.
  • Figure 20 (A) shows the T6RB dissociation constant ( K d ) curve and its value
  • Figure 20 (B) shows the circular dichroism (CD) spectrum of T6RB in the absence/presence of potassium ions (K + )
  • Figure 20 (C) shows the fluorescence intensity (FI) of NMM bound to T6RB in the absence/presence of K +
  • (D) in Figure 20 shows the results of the biomarker topology analysis.
  • Figure 21 shows parameter optimization results for TSA reaction.
  • Figure 21 (A) is aptamer concentration
  • Figure 21 (C) is H 2 O 2 concentration
  • Figure 21 (D) is TSA. Shows optimization results according to reaction time.
  • Figure 22 shows the evaluation results of T6RB aptamer.
  • ELONA and ELONA equipped with TSA are compared and analyzed.
  • Figure 23 shows the results confirming CRC sEV detection by TSA under optimized conditions.
  • Figure 23 (A) shows the sensitivity of SW620 sEV
  • Figure 23 (B) shows the sensitivity of LS 174T sEV
  • Figure 23 (C) shows the sensitivity of HT29 sEV
  • Figure 23 (D) shows CRC, HS and specificity of T6RB for sEVs derived from normal colon.
  • SW620 human colon cancer
  • LS 174T human colon cancer
  • CCD-18Co human normal colon
  • SKBR3 human breast cancer
  • U-87 MG human glioblastoma
  • cells were replaced with conditioned medium supplemented with 5% (v/v) sEV-depleted FBS (Gibco; Thermo Fisher Scientific) and 1% (v/v) penicillin-streptomycin and incubated for 48 h. Cultured in a 5% CO 2 incubator under humidified conditions at 37°C. The conditioned medium containing the secreted sEVs was recovered, and the sEVs were concentrated, isolated, and purified for further use.
  • sEV-depleted FBS Gibco; Thermo Fisher Scientific
  • penicillin-streptomycin penicillin-streptomycin
  • the conditioned medium was first subjected to a series of centrifugations at 300 xg for 5 min, 2,000 xg for 20 min, and 10,000 xg for 30 min to remove cells, cell debris, microvesicles, and apoptotic bodies, respectively. Afterwards, the supernatant was sequentially syringe filtered using filters with pore sizes of 0.45 and 0.22 ⁇ m (Sartorius, G ⁇ ttingen, Germany) to exclude extracellular vesicles larger than 200 nm in size. For concentration and purification, a tangential flow filtration system with a 300K membrane (Pall Corporation, New York, NY, USA) was used until the medium was concentrated to the desired volume.
  • sEVs were isolated from human serum (HS) (Sigma-Aldrich, St Louis, MO, USA) in the same manner as described above, except that they were diluted in 1 ⁇ PBS before serial centrifugation.
  • HS human serum
  • SW620 positive target
  • HS healthy human serum
  • NTA Nanoparticle Tracking Analysis
  • the concentration and size distribution of isolated sEVs were analyzed using a NanoSight NS300 instrument (Malvern Panalytical, Malvern, UK).
  • the laser was set at 532 nm and three 60-second videos were recorded for each sample at 25 frames per second using an sCMOS camera.
  • the camera level was set to 14 with well-adjusted camera focus and the detection threshold was set to 5. Brownian motion of particles was evaluated using NanoSight software (Malvern Panalytical). NTA settings were kept consistent between samples and samples were diluted in PBS for optimal measurements.
  • Cryo-TEM was used to determine the size of SW620 cells and HS-derived sEVs in TEM images. Before sample application, the grid was glow discharged to make it hydrophilic. Each sEV sample (3 ⁇ L) was placed on a perforated carbon-coated copper grid (200 mesh; Quantifoil, profl ⁇ bichau, Germany) and vitrified using a Thermo Scientific Vitrobot (Thermo Fisher Scientific) by placing it in liquid ethane and maintaining the temperature. liquid nitrogen. After vitrification, the samples were stored in liquid nitrogen. Before imaging, the stored samples were transferred to a cryoholder where the temperature was continuously maintained at approximately -180°C using liquid nitrogen. Cryo-TEM images were obtained at 25 kV using a JEM-2100PLUS electron microscope (JEOL, Tokyo, Japan) coupled with a CMOS camera.
  • JEOL JEM-2100PLUS electron microscope
  • Equal volumes of SW620 cells and HS-derived sEVs were concentrated using an Amicon Ultra-0.5 device with a 30K filter (Merck Millipore, Burlington, MA, USA) according to the manufacturer's instructions. Each sample was supplemented with 5x SDS-PAGE loading buffer (Biosesang, Seongnam, Republic of Korea), heated at 95°C for 10 min, and incubated at 300 V for 10 min for 20 min using the Mini-Protean tetra system (Bio-Rad Laboratories). % TGX stain-free protein gel (Bio-Rad Laboratories, Hercules, CA, USA).
  • an important step in E-SELEX is to efficiently separate the bound and unbound aptamer pools.
  • immunoplates as a facile targeted sEV immobilization platform that can not only maintain the integrity of biologically active sEVs but also facilitate the selection of high-quality aptamers with diagnostic value.
  • oligonucleotides used were synthesized by Integrated DNA Technologies (IDT; Coralville, IA, USA). The types of DNA included in the DNA library and the types of forward and reverse primers used are shown in Table 1 below.
  • a random library was designed with a central random region of 40 nucleotides (N40) between the primer regions at both ends used in the PCR amplification step.
  • SW620 cell-derived sEVs and normal human serum (HS)-derived sEVs were used as positive and counter targets, respectively, and selection was performed using maxi-binding immunoplates throughout the E-SELEX process.
  • a nascent single-stranded DNA (ssDNA) library 100 nM ) was denatured at 95°C for 5 minutes and rapidly cooled on ice for 10 minutes.
  • Negative selection was performed first to remove ssDNA likely to bind to BSA and immune plates. 1 g/L of BSA solution was inoculated into the immune plate and incubated at 37°C for 2 hours. After washing three times with washing buffer (WB; DPBS with 5 mM MgCl 2 ) on a shaker (500 rpm) for 30 seconds each, the prepared ssDNA library was added and incubated for 1 hour. Afterwards, the supernatant containing unbound ssDNA was recovered and concentrated using Nucleospin Gel and PCR clean-up (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions.
  • WB washing buffer
  • DPBS DPBS with 5 mM MgCl 2
  • the recovered ssDNA was then amplified using symmetric PCR (Asy-PCR) with different ratios of primer concentrations. Briefly, mixture buffer for Asy-PCR reactions containing 0.2 mM dNTPs, ssDNA template recovered from each selection, high-fidelity nPfu-forte DNA polymerase, and a forward-to-reverse primer ratio of 20:1 in 1x nPfu forte (Enzynomics , Daejeon, Republic of Korea).
  • the thermal cycling protocol was set up as follows: initial denaturation at 95°C for 3 min, followed by 20 cycles of 10 s at 95°C (denaturation), 1 min at 68°C (annealing and elongation), and a final extension of 5 min at 68°C. .
  • the Asy-PCR product was separated on a 2.5% agarose gel, and the ssDNA band was extracted and purified using Nucleospin gel and PCR clean-up.
  • SW620 sEV (20 ⁇ g/mL) were incubated in immunoplates at 37°C for 2 h and then washed (three times) by WB.
  • ssDNA from negative selection was added to the wells, then incubated at 37°C for 1 hour and washed (three times) with WB to remove unbound ssDNA.
  • samples from each well were suspended in BB and heated to 95°C for 10 min to recover bound ssDNA from sEVs and then processed as previously described.
  • Counter selection which relies on incubation of HS-derived sEVs with ssDNA pools, was also performed in the same manner as negative selection to eliminate ssDNA binding to normal sEVs.
  • stringent conditions were performed in continuous E-SELEX loops as described in [Table 2].
  • AC refers to each step in the E-SELEX loop cycle.
  • Negative selection in the E-SELEX procedure was performed to remove non-target single-stranded DNA (ssDNA) that binds nonspecifically to components of the buffer and/or the immune plate itself. Unbound ssDNA obtained from negative selection was enriched for further selection containing three E-SELEX loops, where each loop (denoted A-, B-, and C-loop) was used for four consecutive positive selections and one It included one counter-screening. Considering the high metastatic potential, SW620 cell-derived sEVs were selected as a positive selection target, and HS-derived sEVs were selected as a counter-selection target to establish a clinical environment for potential in vivo application. The loop was repeated three times for a total of 15 individual selection steps, during which the stringency conditions were gradually increased to improve the binding affinity and specificity of the aptamer (Table 2).
  • E-SELEX Another important consideration for the success of E-SELEX is monitoring the E-SELEX procedure to determine the number of loops performed.
  • qPCR can study DNA pools of diverse sequences and provides important information about the homogeneity of ssDNA recovered through E-SELEX in terms of ssDNA pool diversity.
  • the number of loops was selected by analyzing ssDNA obtained from each loop (A-C) by qPCR.
  • PCR was performed on a mixture containing the ssDNA template recovered from each selection and a forward-reverse primer ratio of 1:1 in 1X TOPreal qPCR premix (Enzynomics).
  • the thermal cycling protocol was set up as follows: initial denaturation at 95°C for 15 min, followed by 20 cycles of 10 s at 95°C (denaturation), 15 s at 60°C (annealing), and 15 s at 72°C (extension). This was followed by melting temperature (Tm) analysis while gradually increasing the temperature from 55°C to 95°C at an increment rate of 0.5°C.
  • the initial library of ssDNA is highly heterogeneous in sequence due to a random region of 40 nucleotides. However, heterogeneity will be reduced because an iterative process of selecting a pool of aptamers that specifically bind to the target will be continuously performed. This expected behavior was demonstrated through changes in amplification and melting curves ( Figure 4).
  • amplification of the random library red line; Lib
  • amplification curves were analyzed quantitatively by calculating the ratio of the fluorescence intensity in the last cycle ( 20th ) to the maximum fluorescence intensity in the final qPCR cycle.
  • a ratio higher than 0.95 was considered a plateau of amplification and was sufficient to ensure high homogeneity in ssDNA libraries.
  • the random ssDNA library (Lib) shows the lowest ratio, reflecting significant heterogeneity, but the ratio increases as the E-SELEX loop is continuously repeated.
  • the ratio of C-loop alone showed a value greater than 0.95, proving that C-loop reached a plateau in amplification due to the increase in ssDNA homogeneity.
  • the ssDNA obtained from the C-loop was analyzed by NGS, and the top 10 candidate aptamers were selected based on frequency (%) for further evaluation.
  • NGS Next-generation sequencing
  • the concentrated ssDNA pool was symmetrically PCR amplified with primers to generate dsDNA, resolved on an agarose gel (2.5%), and purified using Nucleospin Gel and PCR clean-up. Finally, the purified dsDNA was processed and the sequence of the candidate aptamer was analyzed through NGS (next-generation sequencing), which was performed by a sequencing company (Clinomics, Ulsan, Republic of Korea).
  • the forward primer of SEQ ID NO: 2 was included in the 5' part of the aptamer sequence
  • the reverse primer of SEQ ID NO: 3 was included in the 3' part of the ssDNA library [Table 3 ], the forward and reverse primer portions are not indicated.
  • CCE stands for colorectal cancer exosome aptamer.
  • Enzyme-linked oligonucleotide assay (ELONA)
  • the immune plate was further incubated with streptavidin-peroxidase polymer (Sigma-Aldrich; 1:1,000) diluted in 0.1% PBST for 1 hour at 37°C and then washed (three times) with 0.1% PBST.
  • streptavidin-peroxidase polymer Sigma-Aldrich; 1:1,000
  • 1x TMB substrate solution (Invitrogen, Carlsbad, CA, USA) was added to each well and incubated for 6 min in the dark and at room temperature.
  • the reaction was terminated by adding 0.5 M sulfuric acid, and the absorbance was immediately measured at 450 nm using a Spectramax iD5 multi-mode microplate reader (Molecular Devices, San Jose, CA, USA).
  • the normalized value ⁇ A was calculated based on the following equation.
  • At is the absorbance of the sample containing both sEV and aptamer
  • Ac is the absorbance of the control containing only sEV.
  • concentration of aptamer varies depending on the analysis method, and all experiments were repeated three times.
  • Primer regions were included in the ssDNA library for PCR amplification during the E-SELEX process. Because shorter length aptamers are synthesized at lower cost and are more suitable for aptamer-based biosensor development, we performed post-SELEX optimization of the CCE-10 aptamer by truncating the forward or reverse primer regions. CCE-10 aptamers lacking the forward, reverse, or both regions were designated CCE-10F, CCE-10R, and CCE-10FR, respectively.
  • the CCE-10 aptamer was indicated by SEQ ID NO: 13 in the above-mentioned Table 3, but in [Table 4] below, it was named CCE 10' including the forward and reverse primer portions, and was listed as SEQ ID NO: 14.
  • the binding affinity of cleaved CCE-10 aptamers (CCE-10F, CCE-10R, and CCE-10FR) and SW620 sEV (positive target) is similar to that of uncleaved CCE-10'.
  • the binding affinity was similar to that of the aptamer.
  • the binding affinity to HS sEV (opposite target) was significantly different. Specifically, aptamers without the forward primer region (CCE-10F and CCE-10FR) had further reduced binding affinity for HS sEVs, whereas aptamers without the reverse primer region (CCE-10R) had a reduced binding affinity. appeared to increase significantly.
  • CCE-10F The binding affinity of CCE-10F was quantitatively evaluated by estimating Kd. Specifically, CCE-10F was exposed to a certain number of SW620 and HS sEVs at various concentrations (0-100 nM) and then analyzed using the ELONA method described above in Example 3.
  • Figure 7 shows the dissociation constant ( K d ) curves of CCE-10F (A) and CCE-10FR (B).
  • K d dissociation constant
  • CCE-10F secondary structure The two-dimensional and three-dimensional structures of CCE-10F secondary structure were predicted and analyzed using OligoAnalyzer Tool (IDT) and Mol * 3D Viewer (RCSB PDB), respectively.
  • IDTT OligoAnalyzer Tool
  • RCSB PDB Mol * 3D Viewer
  • FIG 8 (A) shows that CCE-10F contains two different stem-loop motifs with a minimum Gibbs free energy of -2.39 kcal/mol at 37°C.
  • the affinity of CCE-10F was compared with that of a random library (Lib) and a random library without the forward primer region (Lib-F).
  • Lib random library
  • Lib-F the forward primer region
  • FIG. 8 (D) does not show binding to sEVs derived from CCD-18Co cells (normal colon), SKBR3 cells (human breast cancer), and U-87 MG cells (human glioblastoma), but binds to sEVs derived from SW620 cells. This indicates that CCE-10F has high binding specificity.
  • CCE-10F allows it to detect sEVs derived from CCD-18Co cells, SKBR3 cells, and U-87 MG cells, even though sEVs derived from CCD-18Co cells, SKBR3 cells, and U-87 MG cells were not included as targets of counterscreening. It was confirmed that the affinity of this aptamer for is low and is similar to the binding affinity for HS-derived sEV.
  • aptamer (Apt) alone or Aptamer-SW620 sEV were cultured in 80% human serum (Sigma-Aldrich) for 0–48 h. Samples recovered at each time interval were first treated with proK (200 ⁇ g/mL) at 37°C for 1 hour to remove serum or sEV-derived proteins and stored at -80°C until use. Next, each sample was mixed with Novex TBE-urea sample buffer (Invitrogen), heated at 95°C for 10 min, and dissolved in a urea-modified polyacrylamide gel (15%).
  • Figure 8 (E) shows gel images and stability (%) of aptamers cultured in various time frames from 0 to 48 hours.
  • Aptamer alone and Apt-sEV were stable for up to 24 hours, maintaining stability of 33% and 44%, respectively.
  • no significant differences were observed for up to 3 hours, after which Apt-sEV showed higher stability than the aptamer alone. This may be because the aptamer binds to sEVs, providing more resistance to nucleases in serum. Since the aptamer used in the serum stability analysis is unmodified, modification with functional groups such as phosphothioate bond and 2' fluoro-base increases the resistance of the aptamer to nucleases and improves stability without compromising binding ability. It is expected to be secured.
  • lysates of SW620-derived sEVs were prepared using RIPA lysis buffer supplemented with 1X Halt protease inhibitor cocktail (Thermo Fisher Scientific). sEVs were mixed with lysis buffer supplemented with protease inhibitors, incubated on ice for 20 min and sonicated in a water bath sonicator (SH-2140D, SAE HAN ULTRASONIC, Seoul, Republic of Korea) with 5 cycles of 30 s on/off at 40 kHz. Processed.
  • ProK proteinase K
  • SW620 cell-derived sEVs were treated with 20 ⁇ g/mL proK (Engenomics) for 1 h at 37°C with gentle vortexing every 20 min.
  • Proteinase activity was inhibited by addition of 5 mM phenylmethylsulfonyl fluoride (PMSF) for 10 min at room temperature.
  • PMSF phenylmethylsulfonyl fluoride
  • trypsin-EDTA 0.25% trypsin-EDTA (Welgene) was added to SW620 cell-derived sEVs and incubated at 37°C for 30 min. Trypsin activity was terminated by adding an equal volume of PBS containing 10% FBS. The final composition of each treatment was stored at -80°C until use.
  • the decomposing enzyme activities of proK and trypsin only act on the extravesicular membrane protein of the vesicle, and the intravesicular protein remains unaffected, making it possible to distinguish the location of the biomarker.
  • Using RIPA lysis buffer releases proteins within vesicles from the lumen of sEVs and membrane proteins from the lipid bilayer, exposing them in their original form.
  • Figure 9 shows the results of biomarker topology analysis.
  • I intact sEV
  • II proteinase K (proK) treated
  • III trypsin treated
  • IV RIPA treated sEV results.
  • Figure 9 (I-III) shows that CCE-10F exhibits negligible binding affinity to proK and trypsin-treated sEV.
  • CCE-10F showed comparable binding affinity to RIPA-lysed sEVs compared to intact sEVs ( Figure 9, I and IV).
  • LS 174T cells The viability of LS 174T cells was first assessed after treatment with SW620 cell-derived sEVs, CCE-10F aptamer-sEV complex (Apt-sEV), and CCE-10F aptamer alone (Apt).
  • LS 174T cells The viability of LS 174T cells was tested in the presence of SW620 and aptamer-SW620 sEVs using CCK-8 (Dojindo, Rockville, MD, USA) according to the manufacturer's instructions.
  • Cells (2 x 10 4 cells/well) were inoculated into a 96-well plate. After overnight culture, cells were treated with PBS, SW620, and Aptamer-SW620 sEV for 24 hours at 37°C.
  • the concentrations of sEV and aptamer used in this analysis were 1 x 10 10 particles/mL and 200 nM, respectively.
  • CCK-8 reagent was added to each well and incubated for an additional 2 hours at 37°C. Finally, the absorbance of the sample was measured at 450 nm using a microplate reader, and the sample treated with PBS was considered to have 100% cell viability.
  • sEVs (1 ⁇ 10 particles) were labeled with 100 ⁇ M CFSE (Abcam, Cambridge, UK) and incubated at 37°C on a shaker at 400 rpm for 2 h in the dark.
  • SEC size exclusion chromatography
  • Fractions were collected and stored at -80°C until use.
  • LS 174T cells (2 x 10 5 cells/well) were seeded in a 24-well plate and cultured at 37°C overnight. Next, the cells were replaced with fresh medium and treated with SW620 and Aptamer-SW620 sEV at 37°C for 24 hours.
  • the concentrations of sEV and aptamer used in this analysis were 1 x 10 9 particles and 200 nM, respectively.
  • concentrations of sEV and aptamer used in this analysis were 1 x 10 9 particles and 200 nM, respectively.
  • cells were washed with DPBS, fixed with 4% paraformaldehyde (PFA), and treated with DAPI for nuclear staining.
  • Confocal images were collected on an LSM 900 confocal microscope (Carl Zeiss, Thornwood, NY, USA) and analyzed with Zen software (Carl Zeiss).
  • Figure 11 (A) shows that both sEV and Apt-sEV labeled with CFSE were taken up by LS 174T cells, as evidenced by the green fluorescence signal. Uptake of sEV and Apt-sEV was also quantitatively assessed by flow cytometry. As shown in Figure 11B (left), significant changes in CFSE intensity were observed in LS 174T cells treated with sEV (orange) and Apt-sEV (green) compared to the control (red). Additionally, in Figure 11 (B) (right), the mean fluorescence intensity (MFI) of CFSE was significantly increased in sEV and Apt-sEV, demonstrating effective uptake of sEV by LS 174T cells.
  • MFI mean fluorescence intensity
  • SW620 CRC cell line is originally derived from metastatic lymph nodes and exhibits the ability to metastasize to the liver. Therefore, sEVs derived from SW620 cells may also have metastatic potential, which leads to the development of high invasion and migration abilities in the cells.
  • CCE-10F To explore potential biomarkers for SW620 sEVs specifically targeted by CCE-10F, we selected LS 174T, a CRC cell line with a low metastatic potential, as a model system and demonstrated that CCE-10F inhibits the metastasis of SW620 cell-derived sEVs. We investigated whether it affected the possibility.
  • Wound healing assays were performed to evaluate the effect of sEVs and aptamer-conjugated sEVs on the migration and proliferation abilities of LS 174T cells. Because sEVs can specifically convey cellular information to the microenvironment surrounding the tumor, we hypothesized that sEVs derived from SW620 cells, which have a high metastatic potential, induce cell proliferation, migration, and ultimately metastasis through the sEV-mediated signaling pathway.
  • LS 174T cells (7 ⁇ 10 5 cells/well) were seeded in 24-well plates and incubated overnight under standard culture conditions. Next, a sterile 200 ⁇ L pipette tip was used to induce wounding by scraping the cell monolayer across the center of the well. Cells were then gently washed twice with DPBS and replaced with conditioned DMEM supplemented with 1% FBS. After treating cells with SW620 or aptamer-SW620 sEV, wound gap images were taken at 0 and 24 hours using an Optinity inverted microscope (MDM Instruments, Seoul, Republic of Korea). Wound gap area was analyzed using ImageJ software (NIH, Bethesda, MD, USA). Wound healing (%) was determined using Equation 2 below.
  • Y wound healing (%)
  • a f is the wound gap area at 24 hours
  • a i is the wound gap area at 0 hours.
  • the concentrations of SW620 sEV and aptamer used in this analysis were 2 x 10 10 particles/ml and 200 nM, respectively.
  • Figure 12 (A) illustrates wound gap images of control cells and cells treated with sEV or Apt-sEV at initial (0 h; upper panel) and final (24 h; lower panel) incubation times.
  • Figure 12 (B) When quantitatively analyzed in Figure 12 (B), it was found that 32% of gap closure was induced in LS 174T treated with sEV alone.
  • SW620 sEV can induce LS 174T migration and proliferation, and that LS 174T, which has a low metastatic potential, can also acquire migration ability.
  • the gap closure of LS 174T treated with Apt-sEV was only 18%, which was significantly reduced compared to the group treated with sEV alone (p ⁇ 0.005).
  • the inhibition of migration and proliferation by Apt-sEVs can be inferred that the interaction between Apt and sEVs blocks the structure of protein-based biomarkers, ultimately preventing downstream cellular responses.
  • transwell migration assay without Matrigel
  • transwell invasion assay with Matrigel
  • Transwell cell migration and invasion assays were performed in 24-well plates using inserts with an 8 ⁇ m polyethylene terephthalate (PET) membrane with a diameter of 6.5 mm (Corning Inc., Corning, NY, USA).
  • PET membranes were precoated with Matrigel (Corning Inc.) prior to cell seeding.
  • LS 174T cells resuspended in serum-free DMEM were seeded in the upper chamber of the insert (5 x 10 4 cells/insert) and treated with PBS, SW620, or aptamer-SW620 sEV.
  • DMEM supplemented with 1% FBS was added to the lower chamber as a chemoattractant.
  • Figures 12 (C) and 12 (D) show that the vertical migration of LS 174T cells was significantly inhibited when Apt-sEV was treated compared to the sEV treatment group (p ⁇ 0.0001).
  • the same inhibitory effect of Apt-sEV was also observed in the transwell invasion assay ( Figures 11E and 11F), and the invasion area (%) between sEV and Apt-sEV treatment groups showed a significant difference (p ⁇ 0.0001).
  • Various biomarkers from previous literature have been identified to be involved in migration and invasion of CRC cell lines, some of which are also involved in angiogenesis, epithelial-mesenchymal transition (EMT) and ultimately tumor metastasis. Additionally, biomarkers of CRC cell-derived sEVs were confirmed to promote CRC liver metastasis.
  • HUVEC cells The viability of HUVEC cells was initially assessed after treatment with SW620 sEV, CCE-10F aptamer-sEV complex (Apt-sEV), and CCE-10F aptamer alone (Apt).
  • Figure 10 (B) confirmed that Apt-sEV (green) and Apt (blue) did not show cytotoxicity to HUVEC cells compared to the control group.
  • statistical analysis confirmed that the cell survival rates of Apt-sEV and Apt groups were not significantly different from those of the control group (one-way ANOVA, p > 0.05).
  • HUVEC cells treated with sEV showed a significant increase in cell survival rate compared to the control group (p ⁇ 0.0001). This suggests that SW620 sEV promotes the proliferation ability of HUVEC through the sEV-mediated signaling pathway.
  • Tube formation assays were performed in 96-well plates (SPL Life Sciences). Before cell seeding, 96-well plates were pre-coated with Matrigel (Corning Inc.) HUVECs suspended in CEFOgro-HUVEC medium containing 1% FBS and seeded on Matrigel-coated plates (1 x 10 cells/well). and treated with PBS, SW620, or Aptamer-SW620 sEV. After 4 hours of incubation at 37°C, cells were photographed using an Optinity inverted microscope (MDM Instruments) and analyzed with ImageJ software. It was defined as the relative number of junctions and total branch length of samples treated with sEV and Apt-sEV for samples treated with PBS. The concentrations of SW620 sEV and aptamer used in this analysis were 1 x 10 9 particles/mL and 200 nM, respectively.
  • Figure 13 (A) shows a representative image of tube formation, showing that only sEV treated cells developed tube formation, whereas Apt-sEV treated cells were not effective in forming tubes comparable to the control group. clearly shown.
  • Figures 13 (B) and 13 (C) the relative junction number and total branch length of the sEV group were both significantly greater than those of the Apt-sEV group (p ⁇ 0.0001).
  • the diagnostic validity of the aptamer is that it not only detects CRC cell-derived sEVs at a concentration ranging from 2.0 ⁇ 10 4 - 1.0 ⁇ 10 6 particles/ ⁇ L, and achieves a detection limit of 1.0 ⁇ 10 3 particles/ ⁇ L, but also detects sEVs in healthy serum, normal This was demonstrated by distinguishing between normal sEVs and non-target sEVs derived from cells and other cancer cells.
  • SW620 sEV biomarkers were closely investigated by systematic analyzes including cell survival, cell uptake, wound healing, transwell cell migration/invasion, and tube formation assays.
  • the biomarkers targeted by CCE-10F promote cell migration, invasion, and angiogenesis through sEV-mediated intercellular communication and may be responsible for additional tumor metastasis processes such as EMT. Therefore, it is expected that the DNA aptamer according to the present invention can be used for diagnosis and treatment in the field of colon cancer liquid biopsy.
  • SW620 human colon cancer
  • LS 174T human colon cancer
  • HT29 human colon cancer
  • CCD-18Co human normal colon
  • cells were replaced with conditioned medium supplemented with 5% (v/v) sEVs-depleted FBS (Gibco; Thermo Fisher Scientific) and 1% (v/v) penicillin-streptomycin and incubated for 48 h. Cultured in a 5% CO 2 incubator under humidified conditions at 37°C. The conditioned medium containing the secreted sEVs was recovered, and the sEVs were concentrated, separated, and purified for further use.
  • sEVs-depleted FBS Gibco; Thermo Fisher Scientific
  • penicillin-streptomycin penicillin-streptomycin
  • the conditioned medium was first subjected to a series of centrifugations at 300 xg for 5 min, 2,000 xg for 20 min, and 10,000 xg for 30 min to remove cells, cell debris, microvesicles, and apoptotic bodies, respectively. Afterwards, the supernatant was sequentially syringe filtered using filters with pore sizes of 0.45 and 0.22 ⁇ m (Sartorius, G ⁇ ttingen, Germany) to exclude extracellular vesicles larger than 200 nm in size. For concentration and purification, a tangential flow filtration system with a 300K membrane (Pall Corporation, New York, NY, USA) was used until the medium was concentrated to the desired volume.
  • sEVs were isolated from human serum (HS) (Sigma-Aldrich, St Louis, MO, USA) in the same manner as described above, except that they were diluted in 1 ⁇ PBS before serial centrifugation.
  • HS human serum
  • SW620 and HS-derived small extracellular vesicles Characterization of SW620 and HS-derived small extracellular vesicles (sEVs)
  • the integrity and sEV markers of sEVs isolated from SW620, LS174T, HT29 (positive target) cells and healthy human serum (HS, counter target) were assessed using NTA, cryo-TEM, and Western blot analysis.
  • NTA Nanoparticle Tracking Analysis
  • MONO Zetaview (PMX-130, Particle Metrix, Bavaria, Germany) was used in light scatter mode to determine the concentration and size of sEV (small extracellular vesicle; hereinafter sEV) samples.
  • sEV small extracellular vesicle
  • the instrument Prior to NTA analysis of samples, the instrument was calibrated using 100 nm polystyrene standard beads (diluted 1:250,000 in deionized water). sEV samples were diluted to 4 - 6 x 10 7 particles/mL using 1x PBS before applying to the instrument for measurement.
  • detailed parameters for NTA analysis were consistently set to minimum brightness of 30, sensitivity of 80, shutter of 100, and cell temperature of 23°C. Data from this experiment was analyzed using ZetaView analysis. Deionized water and PBS used in the analysis were filtered with a 0.1 ⁇ m syringe filter membrane for optimal measurement.
  • Figures 14 (A) to 14 (D) show the characteristics of sEVs analyzed according to this example.
  • Figure 14 (A) is the NTA result for SW620 sEV (insert image: cryo-TEM image)
  • Figure 14 (B) is the NTA result for LS 174T sEV (insert image: cryo-TEM image)
  • Figure 14 (C) NTA results for HT29 sEV (inset image: cryo-TEM image).
  • Figure 14 (D) shows the NTA results for HS sEV (insert image: cryo-TEM image). In each cryo-TEM image, scale bar: 100 nm.
  • Equal volumes of SW620, LS 174T, HT29 cells and HS-derived sEVs were concentrated using a qEV enrichment kit (Izon Science) according to the manufacturer's instructions. Each sample was supplemented with 5x SDS-PAGE loading buffer (Biosesang, Seongnam, Republic of Korea), heated at 95°C for 10 min, and incubated at 300 V for 10 min for 20 min using the Mini-Protean tetra system (Bio-Rad Laboratories). % TGX stain-free protein gel (Bio-Rad Laboratories, Hercules, CA, USA).
  • SW620 sEV, LS 174T sEV, HT29 sEV, and HS sEV were about 100 nm to 150 nm, which was consistent with the value obtained using cryo-TEM. Additionally, SW620, LS 174T, HT29 and HS-derived sEVs all showed circular morphology and were within a size range consistent with previous literature, confirming that sEVs were effectively isolated using the SEC method.
  • an important step in TEV-SELEX is to efficiently separate the bound and unbound aptamer pools.
  • immunoplates as a facile targeted sEVs immobilization platform that can not only maintain the integrity of biologically active sEVs but also facilitate the selection of high-quality aptamers with diagnostic value.
  • oligonucleotides used were synthesized by Integrated DNA Technologies (IDT; Coralville, IA, USA). The types of DNA included in the DNA library and the types of forward and reverse primers used are shown in Table 5 below.
  • a random library was designed with a central random region of 40 nucleotides (N40) between the primer regions at both ends used in the PCR amplification step.
  • sEVs derived from three different CRC cells SW620, LS 174T, HT29
  • HS sEVs derived from three different CRC cells (SW620, LS 174T, HT29) and HS were used as positive and counter targets, respectively, and TEV-SELEX was performed by a known method with some modifications.
  • ssDNA single-stranded DNA
  • Negative selection was performed first to remove ssDNA likely to bind to BSA and immune plates. 1 g/L of BSA solution was inoculated into the immune plate and incubated at 37°C for 2 hours. After washing three times with washing buffer (WB; DPBS with 5 mM MgCl 2 ) on a shaker (500 rpm) for 30 seconds each, the prepared ssDNA library was added and incubated for 1 hour. Afterwards, the supernatant containing unbound ssDNA was recovered and concentrated using Nucleospin Gel and PCR clean-up (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions.
  • WB washing buffer
  • DPBS DPBS with 5 mM MgCl 2
  • the recovered ssDNA was then amplified using asymmetric PCR (Asy-PCR) with different ratios of primer concentrations. Briefly, mixture buffer for Asy-PCR reactions containing 0.2 mM dNTPs, ssDNA template recovered from each selection, high-fidelity nPfu-forte DNA polymerase, and a forward-to-reverse primer ratio of 20:1 in 1x nPfu forte (Enzynomics , Daejeon, Republic of Korea).
  • the thermal cycling protocol was set up as follows: initial denaturation at 95°C for 3 min, followed by 20 cycles of 10 s at 95°C (denaturation), 1 min at 68°C (annealing and elongation), and a final extension of 5 min at 68°C. .
  • the Asy-PCR product was separated on a 2.5% agarose gel, and the ssDNA band was extracted and purified using Nucleospin gel and PCR clean-up.
  • each positive target (sEV, 20 ⁇ g/mL) was incubated in an immune plate at 37°C for 2 h and then washed (three times) by WB. Then, ssDNA from negative selection was added to the wells, then incubated at 37°C for 1 hour and washed (three times) with WB to remove unbound ssDNA. Next, samples from each well were suspended in BB and heated to 95°C for 10 min to recover bound ssDNA from sEVs and then processed as previously described.
  • Negative selection in the TEV-SELEX procedure was performed to remove non-target single-stranded DNA (ssDNA) that non-specifically binds to the buffer and/or components of the immune plate. Unbound ssDNA from negative selection was enriched for further selection containing three E-SELEX loops, where each loop (SW620 sEV, LS 174T sEV, HT29 sEV, HS sEV; 1 st -8 th loop in that order) ) included three consecutive positive screenings and one counterscreening.
  • TEV-SELEX Another important consideration for the success of TEV-SELEX is monitoring the TEV-SELEX procedure to determine the number of loops performed.
  • qPCR can study DNA pools of diverse sequences and provides important information about the homogeneity of ssDNA recovered through TEV-SELEX in terms of ssDNA pool diversity. The number of loops was selected by analyzing ssDNA obtained from each loop (1 st -8 th ) by qPCR.
  • the initial library of ssDNA is highly heterogeneous in sequence due to a random region of 40 nucleotides. However, heterogeneity will be reduced because an iterative process of selecting a pool of aptamers that specifically bind to the target will be continuously performed. This expected behavior was demonstrated through changes in amplification and melting curves ( Figure 16).
  • Figure 16 (A) amplification of the random library (red line; Lib) shows an initial increase and reaches maximum fluorescence intensity, but while the primer region is completely hybridized, the random region in the center partially achieves non-complementary hybridization, making it unstable. A rapid decrease in fluorescence occurred due to the formation of heteroduplex.
  • amplification curves were analyzed quantitatively by calculating the ratio of the fluorescence intensity in the last cycle ( 20th ) to the maximum fluorescence intensity in the final qPCR cycle.
  • a ratio higher than 0.95 was considered a plateau of amplification and was sufficient to ensure high homogeneity in ssDNA libraries.
  • the random ssDNA library (Lib) shows a low ratio, reflecting significant heterogeneity, but the ratio increases as the TEV-SELEX loop is continuously repeated. Only the 8 th -loop (light pink line) showed a fluorescence ratio greater than 0.95, proving that the 8 th -loop reached a plateau in amplification due to the increase in ssDNA homogeneity.
  • the ssDNA obtained in the 8th cycle of Example 2 was analyzed by NGS, and the top 10 candidate aptamers were selected based on frequency (%) for further evaluation.
  • NGS Next-generation sequencing
  • the concentrated ssDNA pool from the final loop ( 8th ) of TEV-SELEX was symmetrically PCR amplified with primers to generate dsDNA, resolved on an agarose gel (2.5%), and using Nucleospin Gel and PCR clean-up. Purified (Macherey-Nagel). Finally, the purified dsDNA was processed and the sequence of the candidate aptamer was analyzed through NGS (next-generation sequencing), which was performed by a sequencing company (Clinomics, Ulsan, Republic of Korea).
  • dsDNA was fragmented with Frag enzyme (MGI, Shenzhen, China) for paired-end (PE) 150 sequencing according to the manufacturer's instructions (MGI FS DNA library preparation set).
  • the fragmented DNA was further selected between 300 and 500 bp by DNA clean beads (MGI), and then recovered to generate a blunt-end and modified to have a single adenosine residue at the 3' end.
  • An adapter sequence with a 5' single thymine residue was ligated to both ends of the DNA fragment.
  • the ligation product was then amplified for 7 cycles and subjected to the following single-strand circularization process:
  • PCR product was heat denatured with a special reverse complementary molecule, a single-stranded circular DNA library was created using DNA ligase, and the remaining linear DNA was digested with exonuclease.
  • the DNA library was finally sequenced using DNBSEQ-T7 (MGI) with a PE read length of 150 bp and evaluated by FastQC (v0.11.8) to assess the overall sequencing quality of the MGI sequencing platform.
  • the forward primer of SEQ ID NO: 2 was included in the 5' part of the aptamer sequence, and the reverse primer of SEQ ID NO: 3 was included in the 3' part of the ssDNA library [Table 7 ], the forward and reverse primer portions are not indicated.
  • T stands for Toggle.
  • candidate aptamers modified with 5'-biotin groups were prepared and then tested against SW620, LS 174T, HT29 (positive target) and HS (counter target) sEVs. Binding affinity was assessed using the ELONA method.
  • Enzyme-linked oligonucleotide assay (ELONA)
  • ELONA was performed using a known method with some modifications. Maxi-binding immunoplate (SPL) wells were first coated with each sEV (1 Blocking was performed with a 3% BSA solution in PBS). After washing three times with PBST, the immune plate was incubated with prepared biotin-conjugated aptamer (IDT) in BB at 37°C for 1 hour and then washed three times with 0.1% PBST. The immune plate was further incubated with streptavidin-peroxidase polymer (Sigma-Aldrich; 1:1,000) diluted in 0.1% PBST for 1 hour at 37°C and then washed (three times) with 0.1% PBST.
  • IDT biotin-conjugated aptamer
  • 1x TMB substrate solution (Invitrogen, Carlsbad, CA, USA) was added to each well and incubated for 6 min in the dark and at room temperature. The reaction was terminated by adding 0.5 M sulfuric acid, and the absorbance was immediately measured at 450 nm using a Spectramax iD5 multi-mode microplate reader (Molecular Devices, San Jose, CA, USA). The normalized value ⁇ A was calculated based on the following equation 3.
  • a t is the absorbance of the sample containing both sEV and aptamer
  • a b is the absorbance of the blank. The concentration of aptamer varies depending on the analysis method, and all experiments were repeated three times.
  • the 10 candidate aptamers with the highest frequency (%) showed different affinities for SW620, LS 174T, HT29, and HS sEVs.
  • T6 affinity was highest for SW620, LS 174T, and HT29 sEVs, and affinity was lowest for HS sEVs.
  • these results confirmed the high binding affinity and specificity of T6 for SW620, LS 174T, and HT29 sEVs; Therefore, T6 was selected for optimization after SELEX.
  • T6 aptamers were included in the ssDNA library for PCR amplification during the TEV-SELEX process. Because shorter length aptamers are synthesized at lower cost and are more suitable for the development of aptamer-based biosensors, we performed post-SELEX optimization of the T6 aptamer by truncating the forward or reverse primer regions. T6 aptamers lacking the forward, reverse, or both regions were designated T6F, T6R, and T6FR, respectively, and the derived sequences of T6 with the additional regions removed were designated T6RA T6RB, T6RB1, T6RB2, and T6RB3, respectively (see Table 8). . The T6 aptamer is represented by SEQ ID NO: 23 in the above-mentioned [Table 7].
  • the binding affinities of the cleaved T6 aptamers (T6F, T6R, and T6FR) to SW620, LS 174T, HT29 sEVs (positive target), and HS sEVs (counter target) are comparable to those of the uncleaved T6 aptamer. was similar to the binding affinity ((A) of Figure 18). However, due to the high binding affinity of T6FR for HS sEVs (countertarget), the specificity was low, so we proceeded in the direction of not selecting.
  • the present inventors cut other regions of T6R (FIG. 19 (A) and (C)) to produce T6RA, T6RB, T6RB1, T6RB2 and T6RB3, and then each positive target ( After analyzing the binding affinity to SW620, LS174T, HT29) and the counter target (HS), the ⁇ A value for each cleaved aptamer was compared and evaluated to select the most appropriate aptamer for this study as T6RB. . As shown in Figure 19 (B), the value of the ⁇ A counter target in T6RB did not change, but the value of the ⁇ A positive target increased, showing the highest binding affinity and specificity.
  • T6RB The binding affinity of T6RB was quantitatively evaluated by estimating K d . Specifically, T6RB was exposed to a certain number of SW620, LS 174T, HT29 and HS sEVs at various concentrations (0-100 nM) and then analyzed using the ELONA method described above in Example 5.
  • Figure 20 (A) shows the dissociation constant ( K d ) curve of T6RB.
  • the absorbance signal increased as the concentration of T6RB increased, showing K d values of 3.848 nM, 5.904 nM, and 5.234 nM, respectively, while HS sEV
  • K d values in the low nanomolar range suggested high binding affinity of T6RB for SW620, LS 174T, and HT29 sEVs. Accordingly, T6RB was determined to be a suitable candidate aptamer for diagnostic applications.
  • T6RB at a concentration of 10 ⁇ M was denatured at 95°C for 5 minutes in a buffer containing 10mM Tris/HCl (pH 7.4) and/or 100mM KCl, and then slowly cooled to 25°C to form secondary structures.
  • CD spectra were recorded on a J-810 spectropolarimeter (Jasco, Tokyo, Japan) in the spectral range of 210–300 nm with a scan speed of 50 nm/min and a bandwidth of 1 nm. All measurements were performed in triplicate and averaged, and the background CD spectrum for the corresponding buffer was measured and subtracted from the experimental spectrum.
  • Spectral analysis was performed using Spectra Manager (Jasco), and DNA secondary structure was analyzed using the conformation index r (Equation 4 below).
  • CD 265 and CD 290 are the CD molar ellipticities [ ⁇ ] at 265 nm and 290 nm, respectively.
  • r ⁇ 0.5, 0 ⁇ r ⁇ 0.5, and r ⁇ 0 mainly correspond to parallel, hybrid, and anti-parallel topologies, respectively.
  • Figure 20 (B) shows the circular dichroism (CD) spectrum of T6RB in the absence/presence of potassium ions (K+).
  • the circular dichroism (CD) spectrum of T6RB shows positive peaks in the 210 nm and 265 nm regions, and as a result of calculation based on Equation 2 above, r > 0.5, T6RB It was confirmed that when a G-quadruplex secondary structure is formed, a parallel structure is formed.
  • T6RB at a concentration of 500 nM was denatured at 95°C for 5 min in a buffer containing 10 mM Tris/HCl (pH 7.4) with or without 100 mM KCl, then slowly cooled to 37°C and incubated for an additional 0.5 h. This allowed the secondary structure to be formed. Then, NMM (Cayman Chemical, Ann Arbor, MI, USA) was added to the mixture at a concentration of 5 ⁇ M and incubated for an additional 0.5 h to allow NMM to be inserted into the T6RB structure. Fluorescence intensity (FI) was measured at an excitation wavelength of 399 nm and an emission wavelength of 610 nm on a Spectramax iD5 multimode microplate reader (Molecular Devices).
  • FI Fluorescence intensity
  • lysates of SW620 sEVs were prepared using RIPA lysis buffer supplemented with Halt protease inhibitor cocktail (Thermo Fisher Scientific) at a final concentration of 1X. Briefly, sEVs were mixed with lysis buffer supplemented with protease inhibitors, incubated on ice for 30 min, and then incubated in a water bath sonicator (SH-2140D, SAE HAN ULTRASONIC, Seoul, Republic of) with 5 cycles of 30 s on/off at 40 kHz. Korea) was treated with ultrasonic waves.
  • H-2140D SAE HAN ULTRASONIC, Seoul, Republic of
  • SW620 sEVs were treated with proK (20 ⁇ g/mL, Enzynomics) for 1 h at 37°C with gentle vortexing every 20 min.
  • proK activity was inhibited by treatment with phenylmethylsulfonyl fluoride (PMSF, 5 mM) for 10 min at room temperature.
  • PMSF phenylmethylsulfonyl fluoride
  • trypsin-EDTA trypsin-ethylenediamine tetraacetic acid
  • Figure 20 (D) shows the results of the biomarker topology analysis (in that order, untreated/proteinase K (proK) treated/trypsin treated/RIPA treated sEVs). That is, Figure 20(D) shows that T6RB exhibits negligible binding affinity to proK and trypsin-treated sEVs. Meanwhile, T6RB showed comparable binding affinity to RIPA-lysed sEVs compared to intact sEVs without any treatment ( Figure 20(D)). These results confirmed that the biomarker targeted by T6RB was identified as a protein and was topologically located in the outer membrane of sEV.
  • proK proteinase K
  • TSA with biotin-tyramide (b-tyramide)/SA-PP system was used as an additional step in ELONA to improve the sensitivity of the assay.
  • Experimental conditions optimized for TSA were explored using HT29 sEV (2 ⁇ 10 7 particles/mL) coated on an immune plate, and the concentrations of T6RB, b-tyramide, H 2 O 2 and TSA reaction time were optimized.
  • the first incubation of SA-HRP (1:1000 dilution in PBST) was performed in ELONA under optimized conditions [15 ⁇ g/mL b-tyramide (APExBIO Technology LLC, Texas, USA) in 0.1 M borate (pH 8.5) and 0.001 ⁇ g/mL b-tyramide (APExBIO Technology LLC, Texas, USA). % H 2 O 2 (Sigma-Aldrich)] at 37°C for 25 minutes, followed by a washing step. Finally, a second culture of SA-HRP (1:1000 dilution in PBST) was treated at 37°C for 1 hour followed by a washing step. TMB reactions and absorbance value normalization were handled in the same way as ELONA described above and all experiments were performed in triplicate.
  • T6RB/b-tyramide/H 2 O 2 concentration and reaction time were optimized in TSA, and the results were 160 nM T6RB and 15 ⁇ g/mL, respectively. Results were obtained for b-tyramide, 0.001% H 2 O 2 and a reaction time of 25 minutes ((AD) of FIG. 21).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Hospice & Palliative Care (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un aptamère ADN destiné au diagnostic du cancer colorectal et, spécifiquement, une composition et un kit pour le diagnostic du cancer colorectal, et une composition d'administration de médicament spécifique du cancer colorectal, comprenant chacun l'aptamère constitué des séquences nucléotidiques de SEQ ID N° : 4 à 35. Présentant une affinité et une spécificité élevées pour de petites vésicules extracellulaires (sEV) dérivées de cellules cancéreuses colorectales, l'aptamère de la présente invention peut être utilisé pour le diagnostic spécifique et précis du cancer colorectal. Avec la liaison forte avec des biomarqueurs protéiques spécifiques présents sur la membrane des sEV, l'aptamère ADN supprime le potentiel métastatique du cancer à travers les sEV et est censé trouver des applications dans le traitement du cancer colorectal.
PCT/KR2023/017433 2022-11-02 2023-11-02 Aptamère adn pour le diagnostic ou le traitement du cancer colorectal Ceased WO2024096642A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR1020220144790A KR20240066514A (ko) 2022-11-02 2022-11-02 대장암 진단 및 치료용 dna 압타머
KR10-2022-0144790 2022-11-02
KR1020230150082A KR20250064751A (ko) 2023-11-02 2023-11-02 대장암 진단용 dna 압타머
KR10-2023-0150082 2023-11-02

Publications (1)

Publication Number Publication Date
WO2024096642A1 true WO2024096642A1 (fr) 2024-05-10

Family

ID=90930935

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2023/017433 Ceased WO2024096642A1 (fr) 2022-11-02 2023-11-02 Aptamère adn pour le diagnostic ou le traitement du cancer colorectal

Country Status (1)

Country Link
WO (1) WO2024096642A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118957063A (zh) * 2024-07-22 2024-11-15 宁夏医科大学总医院 一种结直肠癌LncRNA诊断标志物、诊断试剂盒及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109682973A (zh) * 2019-01-02 2019-04-26 中国科学院化学研究所 基于核酸适配体的肿瘤检测方法及试剂盒
KR20190134683A (ko) * 2017-03-30 2019-12-04 어그매니티 나노 엘티디 기능성 올리고뉴클레오타이드의 선택을 위한 방법 및 조성물
CN112501173A (zh) * 2020-11-09 2021-03-16 苏州吉玛基因股份有限公司 一种gpc1 dna适配体及其应用
KR20210112875A (ko) * 2020-03-06 2021-09-15 연세대학교 원주산학협력단 약물이 포획되고 표면에 압타머가 부착된 엑소좀 및 이의 제조방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190134683A (ko) * 2017-03-30 2019-12-04 어그매니티 나노 엘티디 기능성 올리고뉴클레오타이드의 선택을 위한 방법 및 조성물
CN109682973A (zh) * 2019-01-02 2019-04-26 中国科学院化学研究所 基于核酸适配体的肿瘤检测方法及试剂盒
KR20210112875A (ko) * 2020-03-06 2021-09-15 연세대학교 원주산학협력단 약물이 포획되고 표면에 압타머가 부착된 엑소좀 및 이의 제조방법
CN112501173A (zh) * 2020-11-09 2021-03-16 苏州吉玛基因股份有限公司 一种gpc1 dna适配体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHINNAPPAN, R. et al. An integrated lab-on-a-chip platform for pre-concentration and detection of colorectal cancer exosomes using anti-CD63 aptamer as a recognition element. Biosensors and Bioelectronics. published online : 29 October 2022, vol. 220, thesis no. : 114856 (pp. 1-8). *
HUI WANG: "DNase I enzyme-aided fluorescence signal amplification based on graphene oxide-DNA aptamer interactions for colorectal cancer exosome detection", TALANTA, ELSEVIER, AMSTERDAM, NL, vol. 184, 1 July 2018 (2018-07-01), NL , pages 219 - 226, XP093168967, ISSN: 0039-9140, DOI: 10.1016/j.talanta.2018.02.083 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118957063A (zh) * 2024-07-22 2024-11-15 宁夏医科大学总医院 一种结直肠癌LncRNA诊断标志物、诊断试剂盒及应用

Similar Documents

Publication Publication Date Title
WO2010140834A9 (fr) Aptamère d'acide nucléique se liant spécifiquement à des tissus ou à des cellules cancéreuses du pancréas, et utilisation afférente
WO2021086107A1 (fr) Procédé de détermination de la réactivité à un inhibiteur de parp
WO2016018087A1 (fr) Nouveau biomarqueur pour prédire la sensibilité à un agent de ciblage de l'egfr, et son utilisation
WO2024096642A1 (fr) Aptamère adn pour le diagnostic ou le traitement du cancer colorectal
WO2015023146A1 (fr) Méthode de détection de lésions précancéreuses
WO2016036172A1 (fr) Biomarqueur pour prédire la sensibilité à un inhibiteur de protéine kinase et utilisation de celui-ci
WO2015002511A1 (fr) Structure oligonucléotidique améliorée de type nanoparticule présentant une efficacité élevée et son procédé de préparation
WO2010090452A2 (fr) Complexe de petit arn interférence avec une capacité de transmission intracellulaire augmentée
WO2021112464A1 (fr) Dispositif de préparation et procédé de préparation d'un échantillon de biopsie liquide d'exosomes et procédé d'analyse d'échantillon de biopsie liquide d'exosomes préparé par celui-ci
Cha et al. Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell‐Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent
WO2021006570A1 (fr) Procédé de sélection d'aptamères et procédé d'analyse d'immunité utilisant un aptamère
WO2024158242A1 (fr) Composé pour inhiber une mutation de kras g12d et composition pour prévenir ou traiter une maladie cancéreuse, le comprenant en tant que principe actif
WO2021225423A1 (fr) Nouveau ligand d'acide nucléique et son procédé d'identification
WO2018110980A1 (fr) Composition pharmaceutique pour la prévention ou le traitement de l'hépatite b
WO2015108328A1 (fr) Nouveau gène de fusion ntrk1 comme marqueur du cancer colorectal et application associée
WO2017082655A1 (fr) Procédés pour déterminer la résistance à une thérapie anticancéreuse et composition utilisée à cet effet
WO2024177416A1 (fr) Composition pharmaceutique pour le traitement du cancer résistant à la thérapie ciblant l'egfr
WO2015002512A1 (fr) Arnsi spécifique du virus de la dengue, structure d'arn-oligo à double hélice comprenant un arnsi, et composition empêchant la prolifération du virus de la dengue comprenant la structure d'arn
WO2011118994A9 (fr) Gène nlk en tant que marqueur pour le diagnostic du cancer du foie et en tant qu'agent thérapeutique correspondant
WO2023167544A1 (fr) Biomarqueur comprenant la fam167a pour le diagnostic de la résistance contre l'inhibiteur de la tyrosine kinase indépendante de bcr-abl, et composition ciblant la fam167a pour prévenir ou traiter la leucémie myéloïde chronique
WO2022065968A1 (fr) Composition pharmaceutique comprenant de l'évodiamine en tant que principe actif pour la prévention ou le traitement d'un cancer du poumon non à petites cellules
WO2017188707A1 (fr) Nanostructures d'arn de substrat dicer ayant un effet de silençage génique amélioré et leur procédé de préparation
WO2022169342A2 (fr) Composition pharmaceutique destinée à l'inhibition de métastases cancéreuses
WO2018088826A2 (fr) Composition de diagnostic de métastases du cancer colorectal ou de prédiction de pronostic et son utilisation
WO2025249887A1 (fr) Composition pour augmenter la stabilité ou l'expression de l'arn exogène dans les cellules, comprenant un inhibiteur de l'expression ou de l'activité de trim25

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23886336

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE