WO2024096642A1 - Dna aptamer for diagnosis or treatment of colorectal cancer - Google Patents

Abstract

The present invention relates to a DNA aptamer for the diagnosis of colorectal cancer and, specifically, to a composition and a kit for the diagnosis of colorectal cancer, and a colorectal cancer-specific drug delivery composition, each comprising the aptamer consisting of the nucleotide sequences of SEQ ID NOS: 4 to 35. Exhibiting high affinity and specificity for colorectal cancer cell-derived small extracellular vesicles (sEV), the aptamer of the present invention can be used for the specific and accurate diagnosis of colorectal cancer. With the strong binding with specific protein biomarkers present on the sEV membrane, the DNA aptamer suppresses the metastatic potential of cancer through sEV and is expected to find applications in the treatment of colorectal cancer.

Description

DNA aptamer for colon cancer diagnosis or treatment

The present invention is a technology related to a DNA aptamer for colon cancer diagnosis and treatment. Using the aptamer, 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 (CRC), 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. Despite great progress in the prevention and control of colorectal cancer, detection of colorectal cancer and prediction of survival rates are unsatisfactory due to the lack of specific symptoms and accurate biomarkers. Therefore, it is very important to explore colon cancer-specific biomarkers for effective diagnosis of colon cancer.

Meanwhile, small extracellular vesicles (sEVs), also known as exosomes, 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. In particular, 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. For example, the expression patterns of sEVs microRNAs (miRNAs), long non-coding RNAs (lncRNAs), messenger RNAs (mRNAs) and proteins are upregulated in CRC, which increases the diagnostic potential of CRC. Additionally, 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.

In addition, 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). The main advantage of SELEX is that aptamers can be obtained for any biological entity that maintains its natural form, such as cells, tissues, and in vivo models, without prior knowledge of the target.

Accordingly, 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.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

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. In addition, it showed high specificity from non-target sEVs (e.g., sEVs derived from healthy serum and sEVs from normal cells, and sEVs derived from other cancer cells) and effectively distinguished sEVs derived from colon cancer cells. The selected optimal aptamers (SEQ ID NOs: 4 to 17) 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.

In addition, the present inventors constructed Colorectal Cancer (CRC) Toggle sEV-SELEX (hereinafter referred to as TEV-SELEX) to discover high-quality aptamers specific to three types of colon cancer cells (SW620, LS 174T, HT29). . 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. Finally, the best aptamer was obtained through post-SELEX optimization, with K _d values of 3.848 nM, 5.904 nM, and 5.234 nM for SW620, LS 174T, and HT29 cells, respectively, and 3.6 × 10 ² particles/μL, 3.5 × ^Detection limits of 10 ³ particles/μL and 8.4 In addition, it showed high specificity for non-target sEVs (e.g., sEVs derived from healthy serum and sEVs derived from normal colon cells) and effectively distinguished sEVs derived from colon cancer cells with high heterogeneity.

Therefore, 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.

In the present invention, colon cancer is used with the same meaning as “colorectal cancer,” “Colorectal cancer,” and “CRC.”

In the present invention, “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.

In the present invention, “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.

In the present invention, “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.

In one aspect of the present disclosure, 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.

In the present invention, the aptamer can specifically bind to sEVs derived from colon cancer cells. In one embodiment of the present invention, 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.

In the present invention, 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. In the present invention, 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. Illustratively, the sequence of the forward primer may consist of the base sequence of SEQ ID NO: 2, and the sequence of the reverse primer may consist of the base sequence of SEQ ID NO: 3, but are not limited thereto.

In a specific example, 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.

In one aspect, the DNA aptamer may consist of the base sequence of SEQ ID NO: 13. In a specific example, the top 10 candidate aptamers by frequency (%) after SELEX were selected, and aptamers with binding affinity and specificity were selected. As a result, as shown in Figure 6, CCE-10 had the highest affinity for sEV.

Afterwards, optimization of the CCE-10 aptamer sequence was performed by controlling the presence or absence of the forward or reverse primer region. Therefore, as shown in [Table 4], CCE-10, CCE-10F, CCE-10R, and CCE-10FR were used. Therefore, for example, 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. As a result, 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. Additionally, when evaluating the binding affinity of the DNA aptamer, the binding dissociation constant may be 3 to 4 nM, preferably 3.2 to 3.6.

In another aspect of the present invention, the DNA aptamer may consist of the base sequence of SEQ ID NO: 32. In a specific example, the top 10 candidate aptamers by frequency (%) after SELEX were selected, and aptamers with high binding affinity and specificity were selected. As a result, as shown in Figure 17, T6 had the highest affinity for sEVs. Afterwards, 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.

In the present invention, 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.

In another embodiment of the present invention, 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.

In one embodiment, it may be provided in the form of 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.

Illustratively, 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. At this time, the DNA aptamer can be immobilized by any conventional chemical coupling method. For example, biotin is bound to the end of the DNA aptamer to form a complex, and 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.

In addition, various methods 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.

Meanwhile, in one aspect of the present disclosure, 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. For example, 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.

In addition, in one aspect of the present disclosure, 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.

Specifically, the method is

a) generating a random library;

b) performing E-SELEX or TEV-SELEX;

c) analyzing the DNA aptamer sequence through NGS technology; and

d) characterizing the DNA aptamer;

In addition, in the method of producing a DNA aptamer, the 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.

At this time, the method may include contacting the DNA aptamer with 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.

Specifically, when the DNA aptamer is brought into contact with a sample, specific binding occurs between the marker present in sEVs derived from colon cancer cells present in the sample and the DNA aptamer. Therefore, it is possible to detect colon cancer by labeling the DNA aptamer with fluorescence, etc., binding it, and then checking whether a signal is generated.

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. It is a known fact in the art that 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.

In addition, by attaching 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. there is. In addition, 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. siRNA (small interfering RNA) that inhibits the expression of known suicide genes or genes that play an important role in colon cancer growth and metastasis can be directly attached and delivered to sEVs derived from colon cancer cells. The present invention provides the DNA aptamer It may be provided in the form of a colon cancer-specific drug delivery composition containing.

According to the present invention, 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, Hsp90α, 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, and 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 (C) Sensitivity, 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 μm.

Figure 12 shows the results confirming the effect of sEV and Apt-sEV on the migration and invasion of LS 174T. In Figure 12, (A) representative images of wound healing assay at 0 h and 24 h (scale bar: 200 μm), (B) quantitative analysis of gap closure, and (C) representative image of transwell migration assay at 48 h (scale bar: 100 μm), (D) quantitative analysis of migration area, (E) representative image of transwell invasion analysis at 48 hours (scale bar: 100 μm), (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 μm), (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), and (D) in Figure 14 shows the NTA results for HS sEVs (insert: cryo-TEM image). Figure 14 (E) shows the results of Western blot analysis for the expression of CD63, Hsp90α, 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 ), and (C) in Figure 16 is the melt analysis curve. All bars in Figure 16 represent mean ± SD (n=3).

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 ⁺ , and (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 (B) biotin-tyramide (b-tyramide) 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. In Figure 22, 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, and Figure 23 (D) shows CRC, HS and specificity of T6RB for sEVs derived from normal colon.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Example 1]

[Preparation example]

cell culture

SW620 (human colon cancer), LS 174T (human colon cancer), CCD-18Co (human normal colon), SKBR3 (human breast cancer), and U-87 MG (human glioblastoma) cell lines were obtained from the Korea Cell Line Bank (KCLB). . SW620, LS 174T, and CCD-18Co cells were cultured in DMEM (Welgene, Gyeongsan, Korea), while SKBR3 and U-87 MG cells were cultured in RPMI 1640 and MEM, respectively. All cell lines were incubated with 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v/v) penicillin-streptomycin (Welgene) in _humidified conditions at 37°C with 5% CO2. Cultured in each supplemented medium.

Isolation of sEVs

At approximately 80% confluence, 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. 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 μm (Sartorius, Gϕttingen, 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. Next, the concentrated medium was subjected to size exclusion chromatography using a qEV10/35 nm column in a qEV automated fraction collector (Izon Science, Christchurch, NZ) to isolate sEVs in high yield and purity. Fractions containing sEVs from SEC were then pooled and centrifuged again at 5,000 x g for 20 min in a Macrosep with 3K membrane (Pall Corporation) to further concentrate sEVs and then stored in PBS at -80°C until use. did. All centrifugation steps were performed at 4°C to ensure sEV stability. 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.

Example 1-1.

Characterization of SW620 and HS-derived sEVs

The integrity of sEVs and sEV markers isolated from SW620 (positive target) cells and healthy human serum (HS, counter target) were assessed using NTA, cryo-TEM, and Western blot analysis.

Nanoparticle Tracking Analysis (NTA)

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. For all video recordings, 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-transmission electron microscopy (Cryo-TEM)

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 μL) was placed on a perforated carbon-coated copper grid (200 mesh; Quantifoil, Großlϕbichau, 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.

Western blotting

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). Digested proteins were transferred to 0.2 μm PVDF membranes (Bio-Rad Laboratories) in the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories) according to the manufacturer's instructions. Membranes were blanked with 5% BSA TBST solution dissolved in 0.1% TBST (1x TBS with 0.1% Tween-20) and incubated with primary antibody [mouse anti-CD63 (Santa Cruz, Dallas, TX, USA), mouse anti-CD63 (Santa Cruz, Dallas, TX, USA). -CD9, rabbit anti-Hsp90α and rabbit anti-calnexin (ABclonal, Woburn, MA, USA), 1:1,000] followed by incubation with Can Get Signal solution 1 (Toyobo, New York, NY, USA) overnight at 4°C. Washed three times with 0.1% TBST. The membrane was then incubated with the corresponding HRP-conjugated secondary antibodies [HRP goat anti-rabbit IgG (ABclonal), 1:10,000 and HRP goat anti-mouse IgG (Biolegend, San Diego, CA) diluted in Can Get Signal solution 2 (Toyobo). , USA), 1:5000] for 1 hour at room temperature and washed four times with 0.1% TBST. Proteins on the membrane were visualized using Clarity Western ECL substrate (Bio-Rad Laboratories) on a ChemiDoc Imaging System (Bio-Rad Laboratories).

As a result, as shown in Figures 1(A) and 1(B), the sizes of SW620 and HS sEV were 115 and 129 nm, respectively, which matched well with the values obtained using cryo-TEM. Additionally, both SW620- and HS-derived sEVs showed circular morphology and were within a size range consistent with previous literature, confirming that sEVs were effectively isolated using the SEC method. In addition, the Western blot image in Figure 1 (C) confirmed that the isolated sEVs were positive for well-known sEV biomarkers CD63, CD9, and Hsp90α, and negative for calnexin, a marker for the endoplasmic reticulum (ER) membrane. . All these analyzes confirmed that isolated sEV was a suitable target for E-SELEX.

Example 1-2.

In vitro selection of aptamers against SW620-derived sEVs

As can be seen in Figure 2, an important step in E-SELEX is to efficiently separate the bound and unbound aptamer pools. To this end, we used 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.

sEV-SELEX (E-SELEX)

All 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.

Sequence (5'→3') sequence number Random library ATCCAGAGTGACGCAGCA-N ₄₀ -CTGGCTCGAACAAGCTTGC One Forward primer ATCCAGAGTGACGCAGCA 2 Reverse primer GCAAGCTTGTTCGAGCCAG 3

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. Prior _to incubation with target sEVs, 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.

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. . Next, 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. For positive selection, SW620 sEV (20 μg/mL)) were incubated in immunoplates 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. 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. To ensure maximum binding capacity of the aptamer pool targeting only SW620 sEV, stringent conditions were performed in continuous E-SELEX loops as described in [Table 2].

positive screening voice screening Incubation time 1 h (A) ^*
40min (B)
20min (C) 1 h (A)
1.5 h (B)
2 h (C) wash
(30 s, 500 rpm) 3 times (A)
6 times (B)
10 times (C) No washing required Competitor
(Salmon sperm DNA) 0.5 mg/ml (A)
1.0 mg/ml (B)
1.5 mg/ml (C)

^* : AC refers to each step in the E-SELEX loop cycle.

Negative selection in the E-SELEX procedure (Figure 2) 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).

Additionally, the overall process of E-SELEX was simplified by performing ssDNA amplification and generation in one step using Asy-PCR, which does not require magnetic separation, enzymatic digestion, or biotin or phosphate group conjugation for additional NaOH treatment. Asy-PCR using a forward-to-reverse primer ratio of 20:1 under optimized conditions was compared with symmetric PCR with uniform primer concentration through gel electrophoresis analysis. As evidenced by the ssDNA band distinct from the dsDNA band in the results of Figure 3, it was confirmed that ssDNA was generated in the case of Asy-PCR, and the suitability of Asy-PCR was re-proven through the ssDNA generation process.

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.

Quantitative polymerase chain reaction (qPCR)

Selection efficiency was assessed during E-SELEX using 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). In Figure 4A, amplification of the random library (red line; Lib) shows an initial increase and reaches maximum fluorescence intensity, but while the primer region hybridizes completely, the central random region partially undergoes noncomplementary hybridization, resulting in the formation of an unstable heteroduplex. This resulted in a rapid decrease in fluorescence. These results indicate that the low melting temperature (Tm) of 69°C in Figure 4 (B) is consistent with the instability of the heteroduplex. In contrast, the repetitive loops of E-SELEX increased homogeneity by enriching specific ssDNA pools that bind with high binding affinity. Therefore, the ssDNA obtained after C-loop (blue line) showed a normal exponential curve with an extreme plateau during qPCR, and the Tm peak was completely shifted from the lower temperature to the higher temperature of 80.5°C. This means that the proportion of homoduplexes with high stability has increased significantly.

Additionally, to estimate the degree of homogeneity, 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. In this study, a ratio higher than 0.95 was considered a plateau of amplification and was sufficient to ensure high homogeneity in ssDNA libraries. In Figure 5, 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.

All these results from qPCR monitoring showed that the C-loop was optimal to achieve maximum enrichment of the ssDNA pool. Therefore, ssDNA obtained from the C loop was subjected to NGS analysis.

Example 1-3.

Candidate aptamer evaluation

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.

Next-generation sequencing (NGS)

After three E-SELEX loops, 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 sequences of the top 10 candidate aptamers are shown in [Table 3] below.

Sequence (5'→3') ^*# Read Frequency (%) sequence number CCE-1 CCCCGAAGCCTCCTATTCTTAAATTTCCCCATCTTTCTCA 345188 2.18 4 CCE-2 GGCGGAGTAGGGATAAACGGCAGGAAGAGGTGACAGGATG 285719 1.81 5 CCE-3 GTGAGAAAGATGGGGGAAATTTAAGAATAGGAGGCTTCGGG 283314 1.79 6 CCE-4 CCACCGCCGTGAACTAACGTTGGTATTCTCCTCTCCCTTA 225555 1.43 7 CCE-5 CCACCGGCCGACATAGTCATATCTGACCTCTACACTCTCA 200686 1.34 8 CCE-6 CCACATCCCCACAACACCGTCACATCCCCTCATTTCACTG 211848 1.27 9 CCE-7 GACAAAAGAGTTCATGGAAGAAAGGAGAGGAGCCTAGGGT 199772 1.26 10 CCE-8 CCACCCTATCACTTCTTCATTAAACCGTACCCTCCCTACG 185809 1.17 11 CCE-9 CCATCCTGTCACCTCTTCCTGCCGTTTATCCCTACTCCGC 182099 1.15 12 CCE-10 GGCCTAAGAAGGGACAGGGTGACAATCAGGGGGGAGAGGGG 181328 1.15 13

In order to PCR amplify the primer region during the E-SELEX process, 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 3 ], the forward and reverse primer portions are not indicated. CCE stands for colorectal cancer exosome aptamer. To select aptamers with high binding affinity and specificity, candidate aptamers modified with 5'-biotin groups were prepared, and then the binding affinities for SW620 (positive target) and HS (countertarget) sEV were measured using ELONA. It was evaluated using the method

Enzyme-linked oligonucleotide assay (ELONA)

Maxi-binding immunoplate (SPL) wells were first coated with sEVs (1 x 10 ⁸ particles) for 2 hours at 37°C and then washed three times with 0.1% PBST (1x PBS with 0.1% Tween-20). Afterwards, the immune plate was blocked with 3% BSA in 0.1% PBST at 37°C for 2 hours and then washed three times with 0.1% PBST. The immune plate was then incubated with the prepared biotin-conjugated aptamer (IDT) in BB at 37°C for 1 h 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. Next, 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 ΔA was calculated based on the following equation.

<Equation 1>

ΔA = At - Ac

Here, At is the absorbance of the sample containing both sEV and aptamer, and Ac is the absorbance of the control containing only sEV. The concentration of aptamer varies depending on the analysis method, and all experiments were repeated three times.

As shown in Figure 6, the top 10 candidate aptamers with high frequency (%) showed different affinities for SW620 and HS sEV. Interestingly, CCE-10, which had the lowest frequency (%), had the highest affinity for SW620 sEVs and the lowest affinity for HS sEVs. This differed from the initial assumption that aptamers with higher frequency (%) would have higher binding affinity than aptamers with lower frequency (%). Additionally, when estimating the ratio of ΔA _SW620 to ΔA _HS , CCE-10 also showed the highest value. Overall, these results confirmed the high binding affinity and specificity of CCE-10 for SW620 sEV; Therefore, CCE-10 was selected for optimization after SELEX.

Example 1-4

Post-SELEX optimization of CCE-10 aptamer

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.

Sequence (5'→3') sequence number Random library ATCCAGAGTGACGCAGCA-N ₄₀ -CTGGCTCGAACAAGCTTGC One Forward primer ATCCAGAGTGACGCAGCA 2 Reverse primer GCAAGCTTGTTCGAGCCAG 3 CCE-10 ATCCAGAGTGACGCAGCAGGCCTAAGAAGGGACAGGGTGACAATCAGGGGGAGAGGGGCTGGCTCGAACAAGCTTGC 14 CCE-10F GGCCTAAGAAGGGACAGGGTGACAATCAGGGGGAGAGGGGCTGGCTCGAACAAGCTTGC 15 CCE-10R ATCCAGAGTGACGCAGCAGGCCTAAGAAGGGACAGGGTGACAATCAGGGGGGAGAGGGG 16 CCE-10FR GGCCTAAGAAGGGACAGGGTGACAATCAGGGGGGAGAGGGG 17

As shown in Figure 6 (B), 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. However, 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. From these results, it was determined that the forward primer region evaluated in this study could participate in the binding of aptamers to HS sEVs and increase the probability of off-target binding, and that removing the forward primer region from CCE-10 could reduce the likelihood of cancer-derived sEVs It was concluded that it would be helpful in distinguishing sEVs from non-targeted sEVs. Since CCE-10F and CCE-10FR exhibit comparable binding affinities to SW620 and HS sEV, the ratio of ΔA _SW620 to ΔA _HS for each truncated aptamer was evaluated to select the most suitable aptamer for this study. did. As shown in Figure 6B, the ratio of CCE-10F was the highest, demonstrating its superiority in terms of binding affinity and specificity.

Examples 1-5.

Determination of the dissociation constant, Kd

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.

Binding affinity analysis

To assess the binding affinity of the selected aptamer CCE-10F, we varied the concentration of the selected aptamer labeled with biotin (0–100 nM) and determined the equilibrium dissociation constant ( K _d ) using the ELONA method described above. . Normalized ΔA values were expressed as a function of aptamer concentration and K _d was determined using the nonlinear regression equation Y = (Bmax Х X)/( K _d + X). where Y is ΔA, Bmax is the maximum ΔA, K _d is the dissociation constant, and X is the aptamer concentration.

As a result, Figure 7 shows the dissociation constant ( K _d ) curves of CCE-10F (A) and CCE-10FR (B). As shown in Figure 7 (A), for SW620 sEVs, the absorbance signal increases as the concentration of CCE-10F increases, showing a K _d value of 3.41 nM, whereas for HS sEVs, the absorbance signal does not change, resulting in K _d The value could not be determined. K _d values in the low nanomolar range suggested high binding affinity of CCE-10F for SW620 sEV. In addition, the K _d value of CCE-10FR for SW620 and HS sEV was measured in the same manner as for CCE-10F. As a result, a K _d of 6.34 nM was detected for SW620 sEV, and a K _d value was detected for HS sEV. It did not work ((B) in Figure 7). Although the affinity of CCE-10F was 1.9 times higher than that of CCE-10FR, both CCE-10F and CCD-10FR were judged to be suitable candidate aptamers for diagnostic and therapeutic applications. CCE-10F, which has a higher binding affinity to SW620 sEV, was selected for further experiments.

Example 1-6

Best Aptamer Evaluation (CCE-10F)

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.

Figure 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. We hypothesized that the presence of two stem-loop motifs would endow CCE-10F with high binding affinity and specificity for SW620 sEV. Next, 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). As shown in Figure 8 (B), CCE-10F has the highest binding affinity to SW620 sEV, which is significantly higher than the random libraries (Lib and Lib-F).

Additionally, the detectability of CCE-10F against SW620 sEV was investigated using the ELONA method. The results shown in (C) of Figure 8 show that the absorbance signal (ΔA) is proportional to the concentration of SW620 sEV (2.0 Х 10 ⁴ - 1.0 Х 10 ⁶ particles/μL) and the linear regression function is Y = 0.01986X + 0.08825 (R ² = 0.9903) is obtained as follows. Additionally, as a result of calculating the limit of detection (LOD) for SW620 sEV, it was derived to be 1.0 x 10 ³ particles/μL. Considering that the concentration of sEV in serum or plasma is in the range of 10 ⁵ particles/μL, this suggests that it is accurate to strongly verify the diagnostic ability of CCE-10F for CRC cell-derived sEV.

Finally, we evaluated the binding specificity of CCE-10F to sEVs derived from normal colon and other cancer cell lines and to sEVs derived from SW620 and HS. Figure 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. The high specificity of 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 stability analysis in human serum

Finally, the stability of aptamer (Apt) alone and/or Apt-sEV in HS (80%) was further evaluated to confirm the potential in vivo use of the aptamer.

To investigate aptamer stability in serum, 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 μg/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%). Gels were stained with GreenStar Nucleic Acid Staining Solution (Bioneer, Daejeon, South Korea) and imaged using the ChemiDoc Imaging System (Bio-Rad Laboratories). Band intensity was analyzed using Image Lab software (Bio-Rad Laboratories). The band intensity of aptamer or aptamer-SW620 sEV at time 0 was considered 100%.

As a result, 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. Interestingly, 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.

These interesting results could pave the way for the development of various aptamer-based biosensors for specific detection of CRC cell-derived sEVs together with various signaling methods such as fluorescence polarization (FP) and surface-enhanced Raman spectroscopy (SERS). It was confirmed that it exists.

Example 1-7.

Topology analysis of aptamer target biomarkers for SW620 sEV

To investigate the biomarker location on SW620 sEVs targeted by CCE-10F, sEVs were subjected to proteinase K (proK), trypsin and RIPA lysis.

For sEV lysis, 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. For proteinase K (proK) treatment, SW620 cell-derived sEVs were treated with 20 μg/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. For trypsinization, 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.

As a result, 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. That is, Figure 9 (I-III) shows that CCE-10F exhibits negligible binding affinity to proK and trypsin-treated sEV. Meanwhile, CCE-10F showed comparable binding affinity to RIPA-lysed sEVs compared to intact sEVs (Figure 9, I and IV). These results confirmed that the biomarker targeted by CCE-10F was identified as a protein and was topologically located in the outer membrane of SW620 sEV.

Examples 1-8.

Cellular uptake of sEV and Apt-sEV

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).

Cytotoxicity assay

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. Next, 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.

As a result, as shown in Figure 10 (A), sEV (orange), Apt-sEV (green), and Apt (blue) did not show cytotoxicity to LS 174T cells. Statistical analysis also confirmed that the cell survival rate in all cases was not significantly different from that of the control group (one-way ANOVA, p > 0.05). Therefore, the experimental conditions used for cell viability analysis were adopted for further experiments.

Cellular uptake assay

The effect of CCE-10F on sEV uptake by LS 174T cells was examined by confocal microscopy before examining whether specific binding of the CCE-10F aptamer to SW620 sEVs inhibits downstream cellular responses induced by sEVs. Evaluated through image and flow cytometry.

sEVs (1 × ¹⁰ particles) were labeled with 100 μM CFSE (Abcam, Cambridge, UK) and incubated at 37°C on a shaker at 400 rpm for 2 h in the dark. To remove excess CFSE, size exclusion chromatography (SEC) using a qEV10/35 nm column on a qEV automated fraction collector was performed according to the manufacturer's instructions. 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. For confocal microscopy analysis, 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). For flow cytometry, cells were separated by trypsinization, washed with DPBS, resuspended in DPBS supplemented with 2% FBS, and analyzed using a CyFlow Cube 6 flow cytometer (Sysmex Corporation, Kobe, Japan). After recording 3 × ¹⁰⁴ events, the results were analyzed using FlowJo software (BD Biosciences, San Jose, CA, USA).

As a result, the confocal image in 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. Interestingly, uptake of Apt-sEV did not differ from that of sEV alone (ns; p > 0.05). These results strongly demonstrate that the molecular interaction of CCE-10F with potential biomarkers of sEVs does not significantly affect cellular uptake, suggesting that the biological function of potential biomarkers is not related to cellular internalization of sEVs. .

Example 1-9.

Wound healing in sEV and Apt-sEV

The 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. 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 Х 10 ⁵ cells/well) were seeded in 24-well plates and incubated overnight under standard culture conditions. Next, a sterile 200 μL 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.

<Equation 2>

Y = 100 - ( A _f /A _i Х 100)

Here, Y is wound healing (%), A _f is the wound gap area at 24 hours, and 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.

As a result, 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. When quantitatively analyzed in Figure 12 (B), it was found that 32% of gap closure was induced in LS 174T treated with sEV alone. These results show that SW620 sEV can induce LS 174T migration and proliferation, and that LS 174T, which has a low metastatic potential, can also acquire migration ability. On the other hand, 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.

It is noteworthy that wound healing was not affected by the uptake rate of sEV and Apt-sEV as shown in the cellular uptake assay (Figure 11). These results indicate that CCE-10F aptamer can specifically bind to proteins of SW620 sEVs that originally promote the migration and proliferation ability of LS 174T cells and inhibit sEV-mediated cell signaling pathways.

Examples 1-10.

Transwell cell migration and invasion by sEV and Apt-sEV

To further evaluate the possible inhibitory effect of Apt on sEV-mediated tumor progression, transwell migration assay (without Matrigel) and transwell invasion assay (with Matrigel) were performed in LS 174T cells treated with sEV and Apt-sEV. performed.

Transwell cell migration and invasion assays were performed in 24-well plates using inserts with an 8 μm polyethylene terephthalate (PET) membrane with a diameter of 6.5 mm (Corning Inc., Corning, NY, USA). For invasion assays, 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. After 48 h of incubation at 37°C, cells were gently washed twice with DPBS, cells on the lower surface of the insert were fixed with 4% PFA, permeabilized with cold 90% methanol/PBS, and 1 dissolved in 25% methanol. % Dyed with crystal violet. After removing residual crystal violet, cells on the upper surface of the insert were removed with a wet cotton swab, the insert was completely air-dried, and photographs were taken using an Optinity inverted microscope (MDM Instruments). Migration and invasion areas were analyzed using ImageJ software (NIH). The concentrations of SW620 sEV and aptamer used in this analysis were 2 x 10 ⁹ particles/mL and 200 nM, respectively.

As a result, 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.

We believe that the biomarkers targeted by CCE-10F in SW620 sEVs will be involved in promoting cell migration and invasion and are promising against CRC, as evidenced by the enhanced inhibitory effect by the specific binding of CCE-10F to sEVs. It was confirmed that it could be a therapeutic target.

Example 1-11.

Tube formation of sEV and Apt-sEV

We also evaluated the anti-angiogenic effect of CCE-10F on angiogenesis of 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). As a result of analyzing cytotoxicity in the same manner as in Example 8, Figure 10 (B) confirmed that Apt-sEV (green) and Apt (blue) did not show cytotoxicity to HUVEC cells compared to the control group. . In addition, 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). In contrast, 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.

As a result, 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. As evident in 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).

Taken together, consistent with the results observed in cell migration and invasion assays (Figure 12), these results demonstrate that CCE-10F can exert anti-angiogenic effects by specific binding to potential biomarkers for SW620 sEVs. It was comprehensively verified and ultimately confirmed to inhibit downstream cell responses.

In conclusion, the designed E-SELEX method was used to screen the top 10 putative aptamers targeting CRC-derived sEVs, and post-hoc analysis revealed that the best aptamer exhibited the highest binding affinity ( K _d = 3.41 nM) and specificity. Tamer CCE-10F was selected. The diagnostic validity of the aptamer is that it not only detects CRC cell-derived sEVs at a concentration ranging from 2.0 Х 10 ⁴ - 1.0 Х 10 ⁶ particles/μL, and achieves a detection limit of 1.0 Х 10 ³ particles/μL, 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. Additionally, the biological activity of SW620 sEV biomarkers was closely investigated by systematic analyzes including cell survival, cell uptake, wound healing, transwell cell migration/invasion, and tube formation assays. We confirmed that 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.

statistical analysis

All statistical analyzes were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Data are expressed as mean ± standard deviation (SD). Statistical differences between two samples and between two or more samples were evaluated using Student's t-test and one-way ANOVA (one-way analysis of variance). p < 0.05, 0.01, 0.001 and 0.0001 are considered significant and are indicated as *, **, *** and ****, respectively, while p > 0.05 indicates not significant (ns). All experiments were performed in triplicate.

[Example 2]

[Preparation example]

cell culture

SW620 (human colon cancer), LS 174T (human colon cancer), HT29 (human colon cancer), and CCD-18Co (human normal colon) cell lines were obtained from the Korea Cell Line Bank (KCLB). SW620, LS 174T, HT29, and CCD-18Co cells were cultured in DMEM (Welgene, Gyeongsan, Korea). All cell lines were incubated with 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v/v) penicillin-streptomycin (Welgene) in _humidified conditions at 37°C and 5% CO2. Cultured in each supplemented medium.

Isolation of small extracellular vesicles (sEV)

At approximately 80% confluence, 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. 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 μm (Sartorius, Gϕttingen, 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. Next, the concentrated medium was subjected to size exclusion chromatography using a qEV10/35 nm column in a qEV automated fraction collector (Izon Science, Christchurch, NZ) to isolate sEVs in high yield and purity. Fractions containing sEVs from SEC were then pooled and centrifuged again for 20 min at 5,000 did. All centrifugation steps were performed at 4°C to ensure sEVs stability. 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.

Example 2-1.

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.

Nanoparticle Tracking Analysis (NTA)

In this example, 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. 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. For all samples, 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 μm syringe filter membrane for optimal measurement.

Cryo-transmission electron microscopy (Cryo-TEM)

To analyze the size and morphology of sEV samples, the samples were subjected to Cryo-TEM equipment. Carbon-coated copper grids (200 mesh; Quantifoil, Großlϕbichau, Germany) were first made hydrophilic by glow-discharge, and then each sEV sample (3 μL) was applied. Then, they were placed in liquid ethane using a Thermo Scientific Vitrobot (Thermo Fisher Scientific), maintained in liquid nitrogen, and then transferred to a cryoholder where the temperature was continuously maintained at approximately -180°C using liquid nitrogen for vitrification. Cryo-TEM images were acquired at 25 kV using a JEM-2100PLUS electron microscope (JEOL, Tokyo, Japan) coupled with a CMOS camera.

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.

Western blotting

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). Digested proteins were transferred to 0.2 μm PVDF membranes (Bio-Rad Laboratories) in the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories) according to the manufacturer's instructions. Membranes were blocked with 5% BSA TBST solution dissolved in 0.1% TBST (1x TBS with 0.1% Tween-20) and incubated with primary antibodies [mouse anti-CD63 (Santa Cruz, Dallas, TX, USA), rabbit anti-CD63 (Santa Cruz, Dallas, TX, USA). - Hsp90α and rabbit anti-calnexin (ABclonal, Woburn, MA, USA), 1:1,000] incubated overnight at 4°C with Can Get Signal solution 1 (Toyobo, New York, NY, USA), followed by 3 with 0.1% TBST. Washed twice. The membrane was then incubated with the corresponding HRP-conjugated secondary antibodies [HRP goat anti-rabbit IgG (ABclonal), 1:10,000 and HRP goat anti-mouse IgG (Biolegend, San Diego, CA) diluted in Can Get Signal solution 2 (Toyobo). , USA), 1:5000] for 1 hour at room temperature and washed four times with 0.1% TBST. Proteins on the membrane were visualized using Clarity Western ECL substrate (Bio-Rad Laboratories) on a ChemiDoc Imaging System (Bio-Rad Laboratories).

As a result, as shown in (A) to (D) of Figure 14, 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. In addition, the Western blot image shown in Figure 14 (E) shows that the isolated sEVs were positive for CD63 and Hsp90α, well-known sEVs biomarkers, and negative for calnexin, a marker for the endoplasmic reticulum (ER) membrane. Confirmed. All these analyzes confirmed that isolated sEVs were suitable targets for E-SELEX.

Example 2-2.

In vitro selection of aptamers against SW620-derived sEVs

As can be seen in Figure 15, an important step in TEV-SELEX is to efficiently separate the bound and unbound aptamer pools. To this end, we used 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.

Toggle sEV-SELEX(TEV-SELEX)

All 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.

Sequence (5'→3') sequence number Random library ATCCAGAGTGACGCAGCA-N ₄₀ -CTGGCTCGAACAAGCTTGC One Forward primer ATCCAGAGTGACGCAGCA 2 Reverse primer GCAAGCTTGTTCGAGCCAG 3

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) and HS were used as positive and counter targets, respectively, and TEV-SELEX was performed by a known method with some modifications.

Throughout the TEV-SELEX process, negative selection was performed initially to remove ssDNA binding to BSA or the Maxi-binding immunoplate itself (SPL Life Sciences, Pocheon, Republic of Korea). Prior _to incubation with target sEVs, 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.

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. . Next, 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.

For positive selection, each positive target (sEV, 20 μg/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.

Counter selection, relying 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 serum sEVs. To obtain a ssDNA pool with high binding affinity and specificity only for positive target sEVs, stringent conditions were applied in each TEV-SELEX loop (Table 6 below).

Strict conditions for TEV-SELEX positive screening voice screening Incubation time 1 h (1 ^st -2 ^nd ) ^*
45min ( ^{3rd -4th} )
30 minutes (5-6 ^days )
20min ( ^7-8th ) 1 h (1 ^st -2 ^nd )
1.25 h ( ^{3rd -4th} )
1.5 h (5-6 ^th )
2 h (7-8 ^th ) wash
(30 s, 500 rpm) 3 Х (1 ^st -2 ^nd )
5 Х (3 ^rd -4 ^th )
7 Х (5-6 ^th )
9 Х (7-8 ^th ) No washing required Competitor
(Salmon sperm DNA) 0.5 mg/ml ( ^{2nd -8th} )

^* : () My numbers refer to the loop order in TEV-SELEX.

Negative selection in the TEV-SELEX procedure (Figure 15) 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. Considering the highly heterogeneous characteristics of colorectal cancer, three types of CRC cell-derived sEVs, SW620, LS 174T, and HT29, were selected as positive targets, and HS-derived sEVs were selected as countertargets to establish a clinical environment for potential in vivo application. decided. The loop was repeated a total of eight times, during which the stringency conditions were gradually increased to improve the binding affinity and specificity of the aptamer (Table 2). Additionally, the overall process of TEV-SELEX was simplified by performing ssDNA amplification and generation in one step using Asy-PCR, which does not require magnetic separation, enzymatic digestion, or biotin or phosphate group conjugation for additional NaOH treatment.

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.

Quantitative polymerase chain reaction (qPCR)

Selection efficiency was assessed during TEV-SELEX using qPCR. In TOPreal qPCR premix (Enzynomics), qPCR buffer at a final concentration of 1X was mixed with the ssDNA template recovered from each selection. qPCR was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories) using the following thermocycling parameters: initial denaturation (activation) at 95°C for 15 min, followed by 10 s at 95°C (denaturation). ), 20 cycles of 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. qPCR amplification curves and Tm were analyzed with CFX Maestro Software (Bio-Rad Laboratories).

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). In 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. These results indicate that the low melting temperature (Tm) of 68.5°C in Figure 16 (C) is consistent with the instability of the heteroduplex. In contrast, the repetitive loops of TEV-SELEX increased homogeneity by enriching specific ssDNA pools that bind with high binding affinity. Therefore, the ssDNA obtained after 8 ^th -loop (light pink line) showed a normal exponential curve with an extreme plateau during qPCR, and the Tm peak completely shifted from the lower temperature to the higher temperature of 81.5°C. This means that the proportion of homoduplexes with high stability has increased significantly.

Additionally, to estimate the degree of homogeneity, 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. In this study, a ratio higher than 0.95 was considered a plateau of amplification and was sufficient to ensure high homogeneity in ssDNA libraries. In Figure 16 (B), 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.

All these results from qPCR monitoring showed that 8 ^th -loops were optimal to achieve maximum enrichment of the ssDNA pool. Therefore, ssDNA obtained from the 8 ^th -loop was subjected to NGS analysis.

Example 2-3.

Candidate aptamer evaluation

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.

Next-generation sequencing (NGS)

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).

Purified 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:

Briefly, the 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 sequences of the top 10 candidate aptamers in this example are shown in Table 7 below.

Sequence (5'→3')*# Read Frequency (%) sequence number T1 ATCCAGAGTGACGCAGCACCTAGAACCCGCACTAACACTCACCACGACTAACACACACCTGGCTCGAACAAGCTTGC 357817 3.23 18 T2 ATCCAGAGTGACGCAGCAACACTGAAGGGAAGGGAGAGAGGAGTGTGGAGGGTAAAACTGGCTCGAACAAGCTTGC 197439 1.78 19 T3 ATCCAGAGTGACGCAGCACCCCCAATCCGCCTATGCTATCTGGCCTCCATCTCTCTGTCTGGCTCGAACAAGCTTGC 188125 1.7 20 T4 ATCCAGAGTGACGCAGCAACACAGACAAGGCGGTAGAGGAGAGGAGAGGAACTGGCCACTGGCTCGAACAAGCTTGC 178936 1.61 21 T5 ATCCAGAGTGACGCAGCAGTGGCCAGTTCTCTCTCCTCTCCTCTCTACCGCCTTGTCTGTGCTGGCTCGAACAAGCTTGC 170334 1.54 22 T6 ATCCAGAGTGACGCAGCAGGGACAAAGGACACAGGTGGGGGGTGTTGGGATCGGGGGTGCTGGCTCGAACAAGCTTGC 159087 1.44 23 T7 ATCCAGAGTGACGCAGCAGCCTCGCCTCTACTAGATCATACCTCCCTTCCCCTCCGCTCTGGCTCGAACAAGCTTGC 147763 1.33 24 T8 ATCCAGAGTGACGCAGCATGCCACGCCTTTATTTTACGTCCTCTCCCACCCTCTCCTCTCTGGCTCGAACAAGCTTGC 146723 1.32 25 T9 ATCCAGAGTGACGCAGCAGACTAACGGTGCAAAAGTGTGGCAAGAGGGAGAGAGGGGGTCTGGCTCGAACAAGCTTGC 145779 1.32 26 T10 ATCCAGAGTGACGCAGCACACCCCCTCTCTCCCTCTTGCCACACTTTTGCACCGTTAGTCTGGCTCGAACAAGCTTGC 136694 1.23 27

In order to PCR amplify the primer region during the TEV-SELEX process, 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. In Table 7 above, T stands for Toggle.

To select aptamers with high binding affinity and specificity, 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. Next, 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 ΔA was calculated based on the following equation 3.

<Equation 3>

ΔA = A _t - A _b

Here, A _t is the absorbance of the sample containing both sEV and aptamer, and 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.

As shown in Figure 17, 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. Overall, 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.

Example 2-4.

Post-SELEX optimization of the T6 aptamer

Primer regions 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].

Sequence (5'→3') sequence number Random library ATCCAGAGTGACGCAGCA-N ₄₀ -CTGGCTCGAACAAGCTTGC One Forward primer ATCCAGAGTGACGCAGCA 2 Reverse primer GCAAGCTTGTTCGAGCCAG 3 T6F GGGACAAAGGACACAGGTGGGGGGTGTTGGGATCGGGGGTGCTGGCTCGAACAAGCTTGC 28 T6R ATCCAGAGTGACGCAGCAGGGACAAAGGACACAGGTGGGGGGTGTTGGGATCGGGGGTG 29 T6FR GGGACAAAGGACACAGGTGGGGGGTGTTGGGATCGGGGTG 30 T6RA ATCCAGAGTGACGCAGCAGGGACAAAGG 31 T6RB CAAAGGACACAGGTGGGGGGGTGTTGGGATCGGGGTGTG 32 T6RB1 ACACAGGTGGGGGGTGTTGGGATCGGGGGTG 33 T6RB2 CAAAGGACACAGGTGGGGGGTGT 34 T6RB3 ACACAGGTGGGGGGTGT 35

As can be seen in Figure 18, 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. More specifically, to quantitatively compare the binding affinities for T6, T6F and T6R, the ratio of ΔA _positive target to ΔA _{counter target} was analyzed, and as a result, T6R showed the highest value ((BD) in Figure 18) ). Overall, these results confirmed the high binding affinity and specificity of T6R for SW620, LS 174T, and HT29 sEVs; Therefore, after final selection of T6R, a more detailed sequence optimization process was performed.

Afterwards, 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 ΔA 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 ΔA _{counter target} in T6RB did not change, but the value of the ΔA _{positive target} increased, showing the highest binding affinity and specificity. In addition, as shown in Figure 19 (C), the additional sequence optimization of T6RB resulted in a lower value for the ΔA _{positive target} , which was judged to be a sequence that reduces binding affinity, and T6RB was finally selected as the optimal sequence. selected.

Example 2-5.

Determination of the dissociation constant, Kd

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.

Binding affinity analysis

To assess the binding affinity of selected aptamers T6RB, the concentration (0–100 nM) of selected aptamers labeled with biotin was varied and the equilibrium dissociation constant ( K _d ) was determined using the ELONA method described above. Normalized ΔA values were expressed as a function of aptamer concentration and K _d was determined using the nonlinear regression equation Y = (Bmax Х X)/( K _d + X). where Y is ΔA, Bmax is the maximum ΔA, K _d is the dissociation constant, and X is the aptamer concentration.

As a result, Figure 20 (A) shows the dissociation constant ( K _d ) curve of T6RB. As shown in Figure 20 (A), in the case of SW620, LS 174T, and HT29 sEV, 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 In the case of , the absorbance signal did not change, so the K _d value could not be determined. 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.

Circular dichroism (CD) measurement

T6RB at a concentration of 10 μM 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).

<Equation 4>

Here, CD ₂₆₅ and CD ₂₉₀ are the CD molar ellipticities [θ] 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+). In the 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.

Fluorescence measurement of N-methyl mesoporphyrin IX (NMM)

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 μM 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).

As can be seen in (C) of Figure 20, consistent with the circular dichroism spectrum results, it was confirmed that the fluorescence intensity of NMM bound to T6RB appears stronger in the presence of potassium ions (K ⁺ ), which means that T6RB It was confirmed again that a G-quadruplex secondary structure was formed.

Topology analysis of aptamer target biomarkers for T6RB sEV (sEV lysis and enzymatic proteolysis of sEV surface proteins)

For sEV lysis, 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. For proteinase K (proK) treatment, SW620 sEVs were treated with proK (20 μg/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. For trypsinization, trypsin-ethylenediamine tetraacetic acid (trypsin-EDTA, 0.25%) was incubated with SW620 sEVs at 37°C for 30 min, and activity was stopped by adding an equal amount of 10% FBS in 1x PBS. The final composition of each treatment was stored at -80°C until use.

As a result, 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.

ELONA with Tyramide Signal Amplification (TSA) function

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 μg/mL b-tyramide (APExBIO Technology LLC, Texas, USA) in 0.1 M borate (pH 8.5) and 0.001 μg/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.

In order to find the optimal conditions for the ELONA method equipped with TSA, key variables (T6RB/b-tyramide/H ₂ O ₂ concentration and reaction time) were optimized in TSA, and the results were 160 nM T6RB and 15 μg/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).

In Figure 22, as a result of comparative analysis of regular ELONA and TSA-equipped ELONA under TSA optimization conditions, the ΔA of regular ELONA was undetectable for positive targets (SW620, LS 174T, HT29 sEVs) on low concentrations of sEVs. In contrast, in the case of ELONA loaded with TSA, the sEV-free control group did not show a significant ΔA value and showed a much larger ΔA for positive targets (SW620, LS 174T, HT29 sEVs). This can be interpreted as a significant increase in the final ΔA value due to signal amplification by TSA compared to regular ELONA, and is expected to ultimately lead to increased sensitivity.

Example 2-6.

Best aptamer evaluation (T6RB)

Finally, the detection potential for SW620, LS 174T, and HT29 sEVs was analyzed using the T6RB and TSA-equipped ELONA methods with high binding/specificity. The results shown in (A) of Figure 23 show that the absorbance signal (ΔA) is proportional to the concentration of SW620 sEV (2.0 Х 10 ⁴ - 1.0 Х 10 ⁶ particles/μL) and the linear regression function is Y = 0.02782X + 0.03430 (R ² = 0.9871) is obtained as follows. In addition, (B) in Fig. 10 shows that the absorbance signal (ΔA) is proportional to the concentration of LS 174T sEV and the linear regression function Y = 0.01075X + 0.03711 (R ² = 0.9850) was obtained as follows, in Figure 23 (C) shows that the absorbance signal (ΔA) is proportional to the concentration of HT29 sEV and the linear regression function Y = 0.03014X + 0.2759 (R ² = 0.9861) was obtained. In addition, the limit of detection (LOD) for each sEV was calculated as follows: 3.6 x 10 ² particles/μL (SW620), 3.5 x 10 ³ particles/μL (LS 174T), 8.4 x 10 ² particles/μL It was derived as (HT29). This suggests that it is accurate to strongly verify the diagnostic ability of T6RB for CRC-derived sEV, considering that the concentration of sEV in serum or plasma is in the range of 10 ⁵ particles/μL.

In conclusion, using the designed TEV-SELEX method, various CRC cell-derived sEVs were adopted to enable broader targeting of CRC with high heterogeneity, and the top 10 putative aptamers were selected and analyzed to determine the highest binding affinity. The best aptamer T6RB was selected, showing specificity ( K _d = 3.848 (SW620), 5.904 (LS 174T), 5.234 (HT29) nM). The diagnostic validity of the aptamer is that ^it has ^detection limits of ^3.6 was demonstrated by detecting and distinguishing between non-target sEVs, which are healthy serum and normal cell sEVs. In addition, T6RB was confirmed to target proteins present in the outer membrane of sEV, and it is expected that it can be used to construct new biomarkers by identifying biomarkers through future research.

Claims (28)

At least one DNA aptamer for diagnosis or treatment of colon cancer selected from the group consisting of base sequences of SEQ ID NOs: 4 to 17. According to paragraph 1, The aptamer is a DNA aptamer that specifically binds to sEV derived from colon cancer cells. According to paragraph 1, The aptamer is a DNA aptamer, further comprising at least one selected from the group consisting of a forward primer and a reverse primer sequence. According to clause 3, The sequence of the forward primer is a DNA aptamer consisting of the base sequence of SEQ ID NO: 2. According to clause 3, The sequence of the reverse primer is a DNA aptamer consisting of the base sequence of SEQ ID NO: 3. According to paragraph 1, The DNA aptamer is a DNA aptamer consisting of the base sequence of SEQ ID NO: 13. According to clause 6, The DNA aptamer consisting of the base sequence of SEQ ID NO: 13 is a DNA aptamer in which at least one of the forward primer base sequence and the reverse primer base sequence is additionally linked. In clause 7, The DNA aptamer is a DNA aptamer consisting of the base sequence of sequence number 15. According to clause 8, The DNA aptamer is a DNA aptamer having a binding dissociation constant of 3 to 4 nM. At least one DNA aptamer for colon cancer diagnosis selected from the group consisting of base sequences of SEQ ID NOs: 18 to 35. According to clause 10, The aptamer is a DNA aptamer that specifically binds to sEVs derived from various colon cancer cells. According to clause 10, The aptamer is a DNA aptamer, wherein the aptamer further includes one or more selected from the group consisting of a forward primer and a reverse primer sequence. According to clause 12, The sequence of the forward primer is a DNA aptamer consisting of the base sequence of SEQ ID NO: 2. According to clause 12, The sequence of the reverse primer is a DNA aptamer consisting of the base sequence of SEQ ID NO: 3. According to clause 10, The DNA aptamer is a DNA aptamer consisting of the base sequence of SEQ ID NO: 32. According to clause 10, The DNA aptamer is a DNA aptamer having a binding dissociation constant of 3 to 6 nM. A composition for diagnosing or treating colon cancer comprising the aptamer of any one of claims 1 to 9. A kit for diagnosing or treating colon cancer, comprising the composition of claim 10. According to clause 11, The kit is any one selected from the group consisting of a diagnostic sensor, RT-PCR kit, competitive RT-PCR kit, real-time RT-PCR kit, DNA chip kit, and protein chip. A composition for diagnosing colon cancer comprising the aptamer of any one of claims 10 to 16. A kit for diagnosing colon cancer, comprising the composition of claim 20. According to clause 20, The kit is any one selected from the group consisting of a diagnostic sensor, RT-PCR kit, competitive RT-PCR kit, real-time RT-PCR kit, DNA chip kit, and protein chip. A method for producing a DNA aptamer according to any one of claims 1 to 16. The method of claim 23, wherein the method a) generating a random library; b) performing E-SELEX or TEV-SELEX; c) analyzing the DNA aptamer sequence through NGS technology; and d) a method comprising characterizing the DNA aptamer. According to clause 23, The method wherein the aptamer specifically binds to sEV derived from colon cancer cells. A method of providing information for diagnosis or treatment of colon cancer using the DNA aptamer of any one of claims 1 to 16. A method for detecting colorectal cancer sEVs-specific surface biomarkers using the aptamer of any one of claims 1 to 16. A colon cancer-specific drug delivery composition comprising the aptamer of any one of claims 1 to 16.

Images