This application claims foreign priority benefit under 35 U.S.C. §119 of Korean Patent Application No. 10-2014-0101686, filed on Aug. 7, 2014 in the Korean Intellectual Property Office, the contents of which are herein incorporated by reference.
1. Field
The present disclosure relates to a composition or a kit that can be used for detection or diagnosis of various diseases.
2. Description of the Related Art
Researches are under way on techniques for early detection of diseases caused by pathogenic bacteria or viruses and their genetic effects. Examples include detection of specific proteins, i.e., surface markers or antigens (Biosensors & Bioelectronics, vol. 34, pp. 12-24, Apr. 15 2012; ACS Nano, vol. 7, pp. 4967-4976, Jun. 25 2013), detection of pathogenic bacteria through culturing (Nature Reviews Gastroenterology & Hepatology, vol. 9, pp. 312-322, Mar. 27 2012), detection of DNAs through PCR using tailored primers (Science, vol. 314, pp. 1464-1467, Dec. 1 2006; Nature Nanotechnology, vol. 8, pp. 369-375, May 5 2013) and detection by attaching onto beads or particles (Biosensors & Bioelectronics, vol. 29, pp. 46-52, Nov. 15 2011).
However, the detection methods targeting surface markers or antigens are limited in terms of time and cost if relevant antigens or antibodies are unavailable (The Medical Clinics of North America, vol. 96, pp. 1067-1078, November 2012) and the detection methods based on culturing are also restricted a lot in terms of time and cost (Nature Nanotechnology, vol. 8, pp. 369-375, May 5 2013).
The DNA-based detection methods are useful since they can be applied in most cases irrespective of the subjects. At present, DNAs are detected by preparing dozens of single-stranded oligoprimers and conducting PCR based on their complementary binding (Science, vol. 314, pp. 1464-1467, Dec. 1 2006; Nature Nanotechnology, vol. 8, pp. 369-375, May 5 2013) or by attaching them onto particles (Biosensors & Bioelectronics, vol. 29, pp. 46-52, Nov. 15 2011). These DNA-based detection methods are problematic in that detection is possible only when the target DNA is single-stranded or manipulated to be single-stranded (Nature Nanotechnology, vol. 8, pp. 369-375, May 5 2013). In addition, since the specific bonding between complementary bases occurs at moderately high temperatures (Nature Nanotechnology, vol. 8, pp. 369-375, May 5 2013), an unwanted result may be obtained due to non-specific binding if temperature is not sufficiently high or if the sample binding is not in an optimized state (Nucleic Acids Research, vol. 40, pp. W205-W208, July 2012)
The present disclosure is directed to providing a composition or a kit capable of detecting and diagnosing various diseases easily and conveniently.
In an aspect, the present disclosure provides a composition for detection or diagnosis of a disease, containing a dTALE (designed transcription activator-like effector) which binds to a disease-causing gene.
In another aspect, the present disclosure provides a kit for detection or diagnosis of a disease, containing a dTALE (designed transcription activator-like effector) which binds to a disease-causing gene.
The present disclosure is advantageous in that the presence of a specific disease in an individual can be detected conveniently using only the TALE (transcription activator-like effector) of the TALEN (transcription activator-like effector nuclease), which have been used only to cleave a specific base sequence in a target gene or to introduce a new gene.
The composition or kit of the present disclosure is advantageous in that the double-stranded DNA can also be detected. The PCR method currently used for detection of a target DNA requires denaturation of a double-stranded DNA to single strands by applying shock (e.g., heat or acid) since the primer can bind only to a single-stranded DNA. In contrast, the composition or kit of the present disclosure is advantageous in that it can bind to the naturally occurring double-stranded DNA as it is.
In addition, since the composition or kit can accurately and quickly bind to abnormal genes in various diseases, the diseases can be diagnosed or detected easily and conveniently. In particular, by designing a TALE to be capable of binding to an abnormal gene specific for a disease to be detected and loading the same on a commercially available kit, the disease can be self-diagnosed easily and conveniently at low cost.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
As used herein, the term “disease” includes any disease wherein the specific gene sequence which causes the disease is known without limitation.
As used herein, the term “disease-causing gene” refers to a gene sequence known to cause or aggravate the corresponding disease.
As used herein, the term “gene” sequence includes any sequence including one or more DNA strand. Specifically, a DNA-RNA hybrid complex sequence may be included as in AIDS. The DNA sequence includes both single-stranded and double-stranded sequences as well as circular DNA or free DNA. They are schematically shown in
As used herein, the term “TALE (transcription activator-like effector)” refers to a protein secreted by pathogenic Xanthomonas bacteria when they infect various plant cells. The protein can bind promoter sequences in the host plant and activate the expression of plant genes that aid bacterial infection or inhibit the expression of plant genes that interfere with the infection (Current Opinion in Plant Biology, vol. 13, pp. 394-401, August 2010; Science, vol. 326, pp. 1501, Dec. 11 2009). Specifically, in the present disclosure, a “TALE domain” may include two or more TALE repeat units, more specifically 2-30 TALE repeat units. If the TALE domain includes less than 2 TALE repeat units, it is difficult to be designed into a structure for detecting a target gene. And, if the TALE domain includes more than 30 TALE repeat units, the TALE domain becomes too large in size for detection using, for example, a nanostructure. In this regard, the TALE domain may include 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more TALE repeat units, or may include 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less or 20 or less TALE repeat units. For example, a TALE domain according to the present disclosure may have a structure as shown in
In the present disclosure, in order to diagnose a specific disease by transporting the TALE domain, for example, into the nucleus, an NLS (nuclear localization signal) domain may be further bound at the N-terminal. It may be unnecessary if the subject of detection is blood or urine.
Also, the TALE domain of the present disclosure may further contain a domain widely known in the art for its detection. An example is shown in
In the present disclosure, the TALE repeat unit consists of 34 amino acids and may specifically bind to a DNA depending on the 12th and 13th amino acid sequences (Science, vol. 326, pp. 1509-1512, Dec. 11 2009; Science, vol. 335, pp. 720-723, Feb. 10 2012). For example, TALE repeat units having the amino acid sequences shown in Table 1 can specifically bind to DNA bases.
In the present disclosure, the term “dTALE (transcription activator-like effector)” refers to a TALE designed to be capable of specifically binding to a predetermined DNA sequence. Specifically, the dTALE of the present disclosure may be designed to be capable of specifically binding to a disease-causing gene known in the art. Any designing method known in the art can be employed without limitation. Specifically, the method disclosed in PLoS One, vol. 6, issue 5, e19722. May 19, 2011 may be used. More specifically, the dTALE of the present disclosure may be obtained by any method known in the art, for example, by synthesizing amino acids based on amino acid sequence information and connecting them or by designing a plasmid vector having DNA sequence information so that it can transcript an amino acid sequence and expressing it using the target DNA sequence information (Michael R. Green & Joseph Sambrook. Molecular Cloning, 4th Edition. 2012; PNAS, vol. 96 no. 18, pp. 10068-10073, Aug. 31 1999; Chem Soc Rev. Vol. 41, pp. 7001-15, Nov. 7 2012.). Also, it can be obtained using a commercially available TALEN kit. More specifically, the dTALE of the present disclosure may be obtained by preparing a TALE that can bind to a target gene on a commercially available TALEN kit and expressing proteins after separating it from the TALEN kit. The preparation and separation of the TALE and expression into proteins can be conducted according to any method known in the art.
In an aspect, the present disclosure provides a composition for detection or diagnosis of a disease, containing a dTALE (designed transcription activator-like effector) domain which binds to a disease-causing gene.
In another aspect, the present disclosure provides a kit for detection or diagnosis of a disease, containing a dTALE (designed transcription activator-like effector) domain which binds to a disease-causing gene.
According to the present disclosure, the presence of a disease-causing gene can be detected conveniently owing to the specific binding ability of the TALE repeat unit.
The disease-causing gene refers to a gene which exhibits difference in a normal person and in a patient and may be any one known in the art. Specifically, in the present disclosure, the disease-causing gene may be HPV16 E6 genes (specifically HPV16 E6-1 gene, HPV16 E6-2 gene and/or HPV16 E6-3 gene) among the HPV (human papillomavirus) genes known to cause cervical cancer, hypermethylation sites (ICMT-HES3 gene and/or TBR1 gene) known to cause prostate cancer, BNC1 methylation sites (BNC1-1 gene and/or BNC1-2 gene) known to cause pancreatic cancer, APC gene (NM—000038.5) known to cause colorectal cancer, H7N9 or H5N1 virus gene (more specifically NA (neuraminidase) gene) of avian influenza virus, VP1 gene of coxsackie A5 virus (coxsackievirus A5 isolate PUMCH5454Jun07 VP1 gene) causing hand, foot and mouth disease or gag gene of HIV (HIV-1 isolate P6B_acute_A1 gag protein (gag) gene) known to cause AIDS. In the examples of the present disclosure, dTALE (designed transcription activator-like effector) domain including each of the 20 TALE repeat units having the amino acid sequences shown in Table 2 were prepared to detect the specific sequences known as disease-causing sequences of the genes described above.
The composition or kit according to the present disclosure may contain 2-30 TALE repeat units. If the composition or kit contains less than 2 TALE repeat units, it is difficult to be designed into a structure for detecting a target gene. And, if it contains more than 30 TALE repeat units, the resulting complex becomes too large in size for detection using, for example, a nanostructure. In this regard, the composition or kit may contain 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more TALE repeat units, or may contain 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less or 20 or less TALE repeat units.
The composition or kit according to the present disclosure may further contain a tag. The dTALE domain unit and the tag may be present in the composition or kit in the form of a complex, and the tag and the dTALE domain unit may bind at a site different from the binding site of the dTALE domain unit with the gene. In the present disclosure, the tag is used to facilitate the detection of the dTALE domain bound to the target DNA and may be any one known in the art which is capable of binding to the amino acid of the dTALE domain without limitation. For example, the tag may be an HIS tag, a CYS tag, a GST tag or a biotin binding tag, although not being limited thereto. Specifically, the biotin binding tag may be avidin, streptavidin, or a biotin binding peptide having an amino acid sequence LAAIPGAGLIGTH.
The composition or kit according to the present disclosure may further contain a detectable labeling agent and the detectable labeling agent may bind to the tag in the complex. The composition or kit according to the present disclosure is advantageous in that the presence or absence of a target DNA can be detected quickly and conveniently since the detectable labeling agent can easily detect the complex of the dTALE domain bound to the target DNA and the tag by binding thereto.
In the present disclosure, the detectable labeling agent may be any one known in the art that can bind to the tag. Specifically, a quantum dot, a magnetic bead nanoparticle, a gold nanoparticle, a fluorescent dye, a fluorescent protein, a nanophosphor or a silicon nanoparticle may be used, although not being limited thereto.
The quantum dot may be one whose surface is made hydrophilic using materials including an amphiphilic material. For example, the amphiphilic material may be one or more selected from a group consisting of MHPC, DPPE-PEG 2000, Ni-NTA and a mixture thereof. The gold nanoparticle refers to a gold particle having a nanometer-sized diameter and is not particularly limited in shape or size. Anyone having shape and size commonly used in the art may be used. For example, the gold nanoparticle may be spherical and have an average particle size of about 2-15 nm. The size of the gold nanoparticle may be defined adequately depending on the shape of the nanoparticle. For example, if the gold nanoparticle is spherical, its diameter is defined as the size. And, if the gold nanoparticle is not spherical, the dimension of the longest axis may be defined as the size. The fluorescent material refers to a substance which allows light-based detection of the target gene. For example, it may be one or more selected from a group consisting of cyanine, rhodamine, Alexa, fluorescein isothiocyanate (FITC), 5-carboxyfluorescein (FAM), Texas Red and fluorescein, although not being limited thereto.
The disease may be cancer, avian influenza, hand, foot and mouth disease or AIDS, and the cancer may be pancreatic cancer, colorectal cancer, cervical cancer or prostate cancer. However, in the present disclosure, the disease is not limited to the above-described diseases. In the present disclosure, the disease includes any disease wherein a gene which exhibits difference in a normal person and in a patient is known.
In the composition or kit according to the present disclosure, the disease-causing gene may contain one or more DNA strand. Specifically, the gene may be one or more selected from a group consisting of a single-stranded DNA, a double-stranded DNA, a circular DNA, a free DNA and a DNA-RNA hybrid.
In another aspect, the present disclosure provides a method for detection or diagnosis of a disease, including treating a sample obtained from an individual with the composition or kit.
As used herein, the term “individual” includes both an individual suspected to have a disease and a normal individual. A disease may be detected or diagnosed by treating a sample obtained from an individual suspected to have the disease individual with the composition or kit of the present disclosure. The same is applied to a normal individual.
As used herein, the term “treating” includes treating a sample obtained from an individual, specifically a sample derived from an individual and isolated from the individual, with the composition or kit.
As used herein, the term “sample” is not limited as long as it is derived from an individual and contains DNA information of the individual. Specifically, it may be blood or urine but is not limited thereto.
In the method for detection or diagnosis of a disease according to the present disclosure, the gene may contain one or more DNA strand.
In the method for detection or diagnosis of a disease according to the present disclosure, the gene may be one or more selected from a group consisting of a single-stranded DNA, a double-stranded DNA, a circular DNA, a free DNA and a DNA-RNA hybrid.
In the method for detection or diagnosis of a disease according to the present disclosure, the disease may be cancer, avian influenza, hand, foot and mouth disease or AIDS, and the cancer may be pancreatic cancer, colorectal cancer, cervical cancer or prostate cancer.
Hereinafter, the present disclosure will be described in detail through examples and test examples. However, the following examples and test examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples and test examples.
TALENs were prepared by targeting the site cleaved by the StuI restriction site (NEB #R0187S) (TGGACGCAAAGGCCTCAAGG) and the site cleaved by the BamHI restriction site (NEB #R3136S) (GCTCGGGGATCCGAATTCT) in the gene sequence of the T7 bacteriophage using EZ-TAL Assembly Kit-CMV-TALEN (System Biosciences #SBI-GE100A-1, USA) (Biological Procedures Online, vol. 15, pp. 3, Jan. 14 2013; PLoS One, vol. 6, issue 5, e19722. May 19, 2011). Specifically, since the TALEN backbone vector contains 1st and 20th units among the 20 units, the synthesis was conducted after diving a total of 18 units, from the 2nd to the 19th TALE repeat units, into 3 tubes. 6 of the 18 units were added to each tube together with the restriction enzyme and ligase. PCR was conducted for each combination of the 6 units to obtain amplified samples (35 cycles of initial denaturation at 9° C. for 2 minutes→denaturation at 95° C. for 20 seconds→annealing at 61° C. for 20 seconds→extension at 72° C. for 30 seconds→extension at 72° C. for 3 minutes). Finally, the 3 groups were treated with the restriction enzyme and ligase and combined to link the 18 units. Then, the 18 units were introduced into the TALEN backbone vector to finally obtain a dTALE domain having the 20 units.
The synthesized TALEN vector was cut to using Sacl (Takara #1078A) and BsaXI (NEB #R0609S) to include the nuclear localization sequence (NLS) and TALE N-terminal and C-terminal sites and blunted using the Quick blunting kit (NEB #E1201S). It was then cut with HindIII (NEB # R0104L), blunted and inserted into AP-treated pET-21 b plasmid (Novagen #69741-3, Novagen, Germany). For purification of the target protein and use as a marker, biotin and a His-tag were inserted at the C-terminal of the TALE domain. The resulting structure is shown in
A TALE protein was synthesized using the TALE protein expression vector and a bacterial expression system. BL21 E. coli including pET-TALE plasmid was added to 3 mL of a medium containing ampicillin and kept at 37° C. overnight. Then, after transferring to 200 mL of an ampicillin-containing medium and further culturing for 5 hours, 140 μL of 1 M IPTG was added and the culture was kept at 20° C. overnight. The culture medium was transferred to a conical tube and the E. coli cells were collected by centrifugation. The cells were depelleted using 5 mL of a buffer (20 mM Tris-CI, 50 mM NaCl, pH 8.0) and sonicated on ice. After removing E. coli debris through centrifugation, TALE proteins were recovered from the supernatant by binding to Ni-beads (Novagen, Germany), which were then washed and diluted. The TALE protein was concentrated using the 10 kDa MWCO Amicon Ultra column (Millipore, Germany). Finally, a functionalized TALE protein for the target gene was acquired.
MHPC (1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine), DPPE-PEG 2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycol)-2000) and Ni-NTA (1,2-dioleoyl-snglycero-3-N-(5-amino-1-carboxypentyl) iminodiacetic acid succinyl nickel salt) were added to a solution of quantum dots (QDs) in chloroform at ratios of 80%, 15% and 5%. After adding 2 mL of DI water to the solution at 80° C., the mixture was heated for 1 hour to remove the chloroform. Then, the QDs (surface-treated with MHPC, PEG and Ni-NTA) dispersed in water were sonicated for 1 hour to obtain a suspension of single particles. The prepared QDs and the TALE protein having the His-tag were mixed and incubated for 1 hour. Similarly, streptavidin-bound QDs and the biotin-bound TALE protein were mixed and incubated for 1 hour.
The full sequence of the 10-3b T7 bacteriophage (Novagen #70548-3) was cleaved with the PpuMI (NEB #R0506S) restriction enzyme. The resulting 7730-bp fragment (14978-22707) was cut by the StuI (NEB #R0187S) restriction enzyme which cuts the 15481 site and the BamHI (NEB #R3136S) which cuts the 20410 site. The result is shown in
As can be seen from
In addition, the TALE sensor synthesized with the QDs was bound to the T7 bacteriophage fragments and electrophoresed on agarose gel. As can be seen from
As shown in Table 2, sensors containing each TALE domain were prepared in the same manner as in Example 1, for the HPV16 E6 gene known to cause cervical cancer, the site where hyper methylation is observed in prostate cancer patients, the H7N9 avian influenza virus, the coxsackie A5 virus VP1 gene causing hand, foot and mouth disease and the HIV gag gene causing AIDS. In Table 2, only the 12th and 13rd amino acids of the 34 amino acids of the TALE repeat unit are shown.
Sandwich targeting type detection test was conducted using the TALE sensors prepared above. For each disease, two or three TALE sensors were selected and one of the TALE sensors containing biotin was bound onto a streptavidin-coated slide glass. After washing off the unbound TALE sensor, solution samples containing target sites were dropped onto the slide glass. After a predetermined time, the unbound samples were washed off. Then, the other TALE sensors except the one used for coating on the slide glass were dropped onto the slide glass and reacted for a predetermined time. The TALE sensors are those attached to QDs that can emit fluorescence signals at the biotin or HIS tag site. After washing off the unbound TALE sensors, fluorescence was observed (
In
Number | Date | Country | Kind |
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10-2014-0101686 | Aug 2014 | KR | national |