HIGH EFFICIENCY APTAMER COMPLEX COMPRISING BRANCHED DNA AND APTAMER, AND USE THEREOF
20210371863 · 2021-12-02
Inventors
Cpc classification
A61K31/713
HUMAN NECESSITIES
A61K31/7088
HUMAN NECESSITIES
C12N15/115
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C12N15/115
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
Abstract
The present invention relates to a highly efficient aptamer complex comprising a branched DNA and an aptamer, and a pharmaceutical use thereof. More specifically, the aptamer complex of the present invention relates to a highly efficient aptamer complex including a Y-shaped DNA as the branched DNA and using vascular endothelial growth factor (VEGF) as a target molecule. The aptamer complex of the present invention and a pharmaceutical composition comprising the same as an active ingredient are expected to be widely used in the medical field since the binding efficiency with the target molecule is more remarkable than that of the conventional aptamer.
Claims
1. An aptamer complex comprising a branched DNA, an aptamer corresponding to a target sequence, and a terminal DNA.
2. The aptamer complex according to claim 1, wherein the branched DNA is a Y-shaped DNA.
3. The aptamer complex according to claim 1, wherein the Y-shaped DNA is any one or more selected from the group consisting of SEQ ID NOs: 1 to 6.
4. The aptamer complex according to claim 1, wherein the protein corresponding to the target sequence is any one or more selected from the group consisting of vascular endothelial growth factor (VEGF), bovine serum albumin (BSA), adenosine triphosphate (ATP), hepatitis C virus (HCV), and human immunodeficiency virus (HIV).
5. The aptamer complex according to claim 1, wherein the aptamer corresponding to the target sequence is any one or more selected from the group consisting of SEQ ID NOs: 7 to 9.
6. The aptamer complex according to claim 1, wherein the terminal DNA is any one or more selected from the group consisting of SEQ ID NOs: 13 and 14.
7. A drug delivery system comprising the aptamer complex of claim 1 as an active ingredient.
8. A pharmaceutical composition for treating an cancer, comprising the aptamer complex of claim 1 as an active ingredient.
9. The pharmaceutical composition for treating an cancer according to claim 8, wherein the cancer is any one or more selected from the group consisting of melanoma, small cell lung cancer, non-small cell lung cancer, brain tumor, liver cancer, thyroid tumor, gastric cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, glioblastoma, cervical cancer, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, esophageal carcinoma, head and neck tumor, mesothelioma, sarcoma, cholangioma, small intestine adenocarcinoma, childhood malignant cancer, and epidermal cancer.
10. A kit for diagnosing a cancer, comprising the aptamer complex of claim 1 as an active ingredient.
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
[0053] Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are only for illustrate the present invention in more detail and the scope of the present invention is not limited to these examples in accordance with the gist of the present invention.
Example 1: Preparation and Physical Analysis of Target Molecular Capture Construct
Example 1-1. Preparation of Constructed Aptamer (CA) Complex
[0054] In this study, a target molecular capture platform was designed using a “constructed aptamer (CA) complex,” which is an aptamer complex based on a Y-shaped DNA structure. The basic structure of the CA is composed of (1) two Y-shaped DNAs (Y-5, Y-3), which are the framework of the entire structure, (2) Y_Vapt, which is a aptamer capable of hybridizing with a Y-shaped DNA protrusion, and (3) Blu_end as DNA for blunt end formation. The Blu_end is a kind of terminal DNA, and was used to block the overhang (a single strand from which unpaired nucleotides protrude) of the Y-shaped DNA. In the CA platform of the present invention, hybridized oligomer aptamers are used for specific interaction and capture with target molecules. A schematic view of the constructed aptamer complex is shown in
[0055] In this example, to capture human VEGF (hVEGF) overexpressed at the tumor site, the CA was prepared into a hairpin structured hVEGF DNA aptamer (Y_Vapt). Specifically, 6 types of Y-shaped branched DNAs (Y01-5, Y02-5, Y03-5, Y01-3, Y02-3, Y03-3), Blu_end, and Y-Vapt were mixed in the correct amount and annealed under the conditions in Table 1. In the case of CA, the generation of the CA is determined according to the ratio between Blu_end DNA and Y-shaped DNA. When various amounts of Blu_end were used to prepare the CA, it was designated as CA_G# (# of generation). hVEGFaptamer was prepared in the same manner as known technology (Lee, J, et al., J. Chem. Mater. 2016, 28, 3961; and Potty, A, et al., Biopolymers 2009, 91, 145). The sequence information of each material is set forth in Table 2, and when there is a modification at the 5′-end, it is indicated in square brackets.
TABLE-US-00001 TABLE 1 Order 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Temperature 95 85 80 75 70 65 60 55 50 45 40 30 20 4 [° C.] Time [min] 5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 5
TABLE-US-00002 TABLE 2 SEQ ID NO Designation Sequence (5′-3′) bp SEQ ID NO: Y01-5 GCTCCTCGCCTGAAGCTCGATGAATAGCGGTCAGATCCGTACCTA 45 1 SEQ ID NO: Y02-5 GCTCCTCGCCTGAAGCTTCGTTCGCAATACGACCGCTATTCATCG 45 2 SEQ ID NO: Y03-5 GCTCCTCGCCTGAAGCTTAGGTACGGATCTGCGTATTGCGAACGA 45 3 SEQ ID NO: Y01-3 ATCCATGCCTAGACTGGCGATAAGTAGCTGTCCGCTCCTCG 41 4 SEQ ID NO: Y02-3 GCTACTTATCGCCAGCATAACGCTTGCTTGTCCGCTCCTCG 41 5 SEQ ID NO: Y03-3 AGCAAGCGTTATGCGTCTAGGCATGGATTGTCCGCTCCTCG 41 6 SEQ ID NO: Y_Vapt AGCTTCAGGCGAGGAGCCCACCCCGTCTTCCAGACAAGAGTGCAGGGGT 64 7 GGCGAGGAGCGGACA SEQ ID NO: Pri_Vapt CCCGTCTTCCAGACAAGAGTGCAGGG 26 8 SEQ ID NO: Phos_Vapt [Phos]TTTTTAAGCTTGTCCGCTCCTCGCCACCCCTGCACTCTTGTCT 75 9 GGAAGACGGGGTGGGCTCCTCGCCTGAAGCTT SEQ ID NO: Y_Sc AGCTTCAGGCGAGGAGCCCACTATTATGGAACCGAATTTTGTTTCATGT 64 10 GGCGAGGAGCGGACA SEQ ID NO: Pri_Sc TATTATGGAACCGAATTTTGTTTCAT 26 11 SEQ ID NO: Phos_Sc [Phos]TTTTTAAGCTTGTCCGCTCCTCGCCACATGAAACAAAATTCGG 75 12 TTCCATAATAGTGGGCTCCTCGCCTGAAGCTT SEQ ID NO: Blu_end TGTCCGCTCCTCG 13 13 SEQ ID NO: FAM_Blu_end [FAM]TGTCCGCTCCTCG 13 14
[0056] In Table 2 above, [Phos] indicated in the sequence indicates that the sequence is phosphorylated, and [FAM] indicates that a fluorescent substance is linked to the sequence.
[0057] The prepared CA was stored at 4° C. until use and confirmed for the generation and size of the construct with polyacrylamide gel electrophoresis (PAGE 6%, stained with SYBR Gold®, 100 V for 45 minutes), and the results are shown in
[0058] As a control for the CA of the present invention, an oligomeric aptamer (OA) complex, an oligomeric scrambled DNA (OSc), an amplified aptamer (AA) complex, an amplified scrambled DNA (ASc), and a constructed scrambled DNA (CSc) were tested. For the OA, an oligomerichVEGFaptamer DNA was prepared by simple annealing. For the CSc, it was prepared by replacing Y_Vapt used for the preparation of CA with Y_Sc, and is a constructed scrambled DNA without VEGF capture ability, which corresponds to a control. The concentrations of aptamers in the OA, AA, and CA remained the same.
Example 1-2. Preparation of Amplified Aptamer (AA) Complex
[0059] According to CircLigase™ protocol, which is the well-known technique, cyclic DNA was obtained using Phos_Vapt and Phos_Sc as templates. More specifically, the phosphorylated modified DNA was mixed with ATP, MgCl.sub.2, and ligase enzyme in a buffer, and reacted at 60° C. for 12 hours. After the enzyme inactivation process was carried out, the reaction was carried out for 30 minutes at 37° C. with 20 U of exonuclease I and 100 U of exonuclease III, and the enzyme inactivation process was carried out again. The synthesized cyclic DNA was purified by DNA PrepMate™-II, and then confirmed by polyacrylamide gel electrophoresis, and the concentration was measured by ultraviolet absorption at 280 nm.
[0060] A “rolling circle amplification (RCA)” process was carried out to prepare the amplified aptamer (AA) complex using the synthesized cyclic DNA, and then F29 polymerase protocol was carried out. Specifically, a buffer including dNTP, BSA, and F29 polymerase was added to the prepared cyclic Phos_Vapt and Pri_Vapt, and mixed well, and then the solution was sufficiently reacted at 30° C. Then, the solution was reacted at 80° C. to inactivate the enzyme, and then cooled slowly. The amplified aptamer complex using Phos_Sc and Pri_Sc was also prepared in the same manner.
Example 1-3. Dynamic Light Scattering (DLS) Analysis of Prepared Aptamers
[0061] Samples were prepared by a method of dissolving CA including 1 nmol OA, 10 pmol primer AA amplified for 4 hours, or 1 nmol aptamer in 50 mL of PBS. The size of each aptamer in the solution was measured using a Zetasizer Nano (Malvern Instruments, Malvern, UK). The measured results are shown in
Example 1-4. Image Analysis of Prepared Aptamers
[0062] Samples for transmission electron microscope (TEM) were prepared by a method of depositing 10 mM CA on a carbon grid coated with an ultrathin carbon film (400 mesh, Ted Pella, Redding, Calif., USA). The grid was dried for one day before measurement, and TEM images were obtained by JEM-1011 (JEOL, Tokyo, Japan).
[0063] Samples for atomic force microscope (AFM) were prepared by treating newly cut mica (Pelco Mica sheets, Ted Pella Corp.) with NiCl.sub.2 and incubating for 10 minutes. The CA was dissolved in PBS including 10 mM NiCl.sub.2, and the dissolved solution was deposited on the washed mica. After reaction for 30 minutes, the mica surface was washed with deionized water and dried. The dried samples were scanned in non-contact mode using VEECO Dimension 3100/Nanoscope V (VECCO).
[0064] The images obtained by measuring the CA (CA_G4) with the TEM and AFM are shown in
Example 1-5. Synchrotron X-Ray Scattering Measurement of Prepared Aptamers
[0065] Samples were prepared into 50 μL of PBS solution, and synchrotron X-ray scattering measurements were carried out on a 4C beamline. All samples were measured at 25° C. and sample-to-detector distances of 4 m and 1 m. Scattering data was collected through a two-dimensional charge-coupled detector (Model Rayonix 2D Mar, USA) using an X-ray radiation source of λ=0.0756 nm with an exposure time of 60 seconds, and the 2D scattering data was averaged circularly with respect to the beam center and normalized by monitoring the intensity of the X-ray beam transmitted through the scintilliation counter located at the back of the sample. Thereafter, the solvent scattering effect was removed from the scattering data by calculating the scattering intensity I (q) of the spherical particles in the solvent medium by an equation according to a known technique (Li, T, et al., Chem. Rev. 2016, online publishing) and treating it with the Guinier equation to extract the rotation radius (Rg) of the DNA complex (Guinier, A, et al., Wiley: New York, 1955). The results of the X-ray scattering measurements are shown in
[0066] A representative X-ray scattering profile of the DNA complex is shown in
Example 1-6. Confirmation of the Stability in Serum of Prepared Aptamers
[0067] To confirm the stability of the low polymer aptamer complex (oligomeric aptamer; OA), amplified aptamer (AA) complex, and constructed aptamer (CA) complex to DNA nuclease in serum, the aptamer complexes were mixed in a 10% fetal bovine serum (FBS) solution and reacted for 3 days in a dark place at 37° C. The stability for the low polymer aptamer complex was analyzed by polyacrylamide gel electrophoresis, and the stability for the amplified aptamer complex and constructed aptamer complex was analyzed by agarose gel electrophoresis. The results are shown in
[0068] The hVEGF capture ability of the OA and CA_G4 according to the degradation rate is shown in
Example 2: Analysis of Biological Effects of Target Molecular Capture Constructs
Example 2-1. Confirmation of Capture Ability of OA, AA, and CA in hVEGF-Containing Solution
[0069] To confirm the hVEGF capture ability of OA, AA, and CA, each prepared sample was mixed with hVEGF solution for 4 hours at room temperature, and the mixed solution was filtered through a PD-10 column (Sephadex G-25, GE Healthcare, Sweden). The amount of non-purified hVEGF was measured by the hVEGF enzyme-linked immunosorbent assay kit (ELISA kit, Komabiotech) according to the manufacturer's guidelines (absorbance measurement at 490 nm by VICTOR3 V™ (Multilabel Counter, Perkin Elmer, Wellesley, Mass., USA)). The hVEGF capture ability of other samples such as OSc, ASc, and CSc was measured in the same way, and the results are shown in
[0070] As a result of the experiment, the OA and CA (CA_G4) had a remarkable capture efficiency, but the AA showed a relatively low capture efficiency. These results indicate that exposure of the reaction site of the DNA structure is very important to efficiently capture the target molecule. The overall structure of the CA is similar to that of the OA. However, the AA forms a highly entangled structure by the RCA process, so the target aptamer is not exposed to hVEGF, and thus, the interaction with the target molecule is low. The hVEGF capture efficiency of the CA was found to be saturated when the aptamer concentration reached 50 pmol (CA_G4=50 pmol aptamer).
Example 2-2. Confirmation of Cytotoxicity of Prepared Aptamer Complexes
[0071] Cytotoxicity of each aptamer complex (OA, AA, and CA) was evaluated using standard MTT assay. First, A549 cells (lung adenocarcinoma cells) were seeded in 96-well plates at a density of 5×10.sup.3 cells/well and incubated in a CO.sub.2 incubator for 24 hours. The cells were treated with various concentrations (0, 250, 500, 1000, 1500, or 2500 nM) of OA, AA, or CA in serum-free medium (DMEM) for 6 hours, and the supernatant was removed and incubated in fresh serum-containing medium (DMEM) for 20 hours. Then, the mixture was replaced with fresh medium including MTT solution (5 mg/mL), and further reacted for 4 hours. The supernatant was removed and DMSO was added to each well to dissolve purple formazan crystals in the cells, and then absorbance was measured at 570 nm using a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, Perkin Elmer, Wellesley, Mass., USA). The measured value was expressed as a relative percentage based on the measured value of the control sample added with medium only. The results of the MTT assay are shown in
[0072] As a result of the experiment, the cell viability was not significantly reduced at an aptamer concentration of 0 to 250 pmol, and thus, all of the tested aptamer complexes were found to be biocompatible.
Example 2-3. Confirmation of Degree of In Vitro Cell Uptake and Internalization
[0073] To evaluate the degree of cell uptake and internalization of CA, CA manufactured with FAM fluorescently labeled Blu_end was prepared and treated with A549 cells. More specifically, A549 cells were seeded on a glass cover slip placed on a 12-well plate at a density of 2×10.sup.4 cells/well and incubated for 18 hours. Thereafter, the culture broth (DMEM) was replaced with fresh serum-free DMEM containing FAM-labeled CA (containing 0.5 nmol FAM), and incubated for an additional 5 hours. After incubation, cold DPBS was added to block cell uptake, and the cells were washed and finally fixed at 4° C. overnight with 10% neutral buffered formalin (NBF). The cells on the cover slip were stained with DAPI and observed with a confocal laser scanning microscope (Modified Zeiss Axio Observer, Z1 epi-fluorescence microscope), and the image results are shown in
[0074] As a result of the experiment, it was confirmed that the green fluorescence signal of the FAM-labeled CA was expressed in the nuclear region overlapping with the DAPI blue fluorescence, so that the cell internalization of the CA was smooth.
Example 2-4. Confirmation of In Vivo Cancer Treatment Effect of Prepared Aptamer Complexes
[0075] All animal experiments in the present invention were carried out with the approval of the POSTECH Biotech Center Ethics Committee. First, A549 cells (1×10.sup.8) were subcutaneously inoculated into the flanks of each female BALB/c-nu/nu mouse, and then the mice were randomly divided into 5 groups (n=3), and OA, AA, CA, or CSc was administered by injection on days 1 and 5. The CSc has the same structure as the CA in vivo, but the DNA sequence is a different aptamer complex.
[0076] The anticancer effect of the administered aptamers on A549 cells was analyzed by measuring the tumor diameter with a caliper. The measured values were converted into tumor volumes using the equation described in Table 3 below.
TABLE-US-00003 TABLE 3 Tumor Volume = a × b.sup.2 × 0.5 Wherein, a is a short dimension, and b is a long dimension.
[0077] After administration of each aptamer, tumor tracking and body weight change in mice were monitored until day 28, and the results are shown in
[0078] More specifically,
[0079] The body weight change of the mice is shown in
Example 2-5. Confirmation of Histological and Chemical Changes in Tumor Tissue
[0080] For immunohistochemical analysis, each aptamer complex (OA, AA, CA, or CSc) was administered to tumor cell-injected mice, and then, the mice were sacrificed on day 4. Tumors were excised and fixed in 10% NBF for 24 hours, and then embedded with paraffin and sliced to a thickness of 4 mm using a Finesse ME microtome. To confirm histological changes of the tumor, tumor sections were stained with hematoxylin and eosin (H & E) and an ×10 magnification image was obtained with an optical microscope (Nikon eclipse 80i, USA).
[0081] In the case of immunohistochemical staining using CD-31 antibody, the sections sliced into a thickness of 4 mm were treated with a blocking solution (containing 5 w/v % BSA) for 15 minutes to prevent non-specific binding, washed several times with PBS, and treated with primary antibody for 1 hour at room temperature. Thereafter, these were treated with goat anti-rabbit IgG-FITC-labeled secondary antibody for 1 hour. Finally, the cover slip was mounted with a mounting medium including DAPI, and stored in a dark place at 4° C. Fluorescence images were obtained with a confocal laser scanning microscope (Modified Zeiss Axio Observer, Z1 epi-fluorescence microscope) at ×40 magnification.
[0082] The image results are shown in