KR20090121573A - Method for immoboilizing proteins on a cognate dna-modified substrate surface - Google Patents

Abstract

PURPOSE: A method for selectively fixing a protein on the surface of a substrate is provided to simplify the fixation in a biochip and biosensor. CONSTITUTION: A method for selectively fixing a protein on the surface comprises: a step of spotting cognate DNA of protein or peptide on a substrate; and a step of dipping the spotted substrate in a protein solution or peptide solution to fix the protein or peptide. A DNA binding domain is fused in the protein or peptide.

Description

Method for Immoboilizing Proteins on a Cognate DNA-Modified Substrate Surface}

The present invention relates to a method of selectively immobilizing a protein on the surface of a substrate, and more particularly, to a method of selectively immobilizing a protein after spotting cognate DNA of the protein to be immobilized on the substrate.

The main advantages of the orientation-regulated protein immobilization are good steric accessibility and improved functionality for active binding sites on the chip surface (Peluso et. al ., Biochem ., 312: 113-124, 2003; Soellner et al ., Chem . Soc ., 125: 11790-11791, 2003). Therefore, directly immobilizing proteins on the active binding sites on the chip surface is an essential procedure in the fabrication of sensitive biosensors.

Protein microarrays formed on gold thin films are advantageous in that protein-protein interactions can be analyzed by surface plasmon resonance (SPR) imaging systems (Smith and Corn , Spectrosc ., 57) . : 320A-332A, 2003; Steiner et al ., Bioanal . Chem ., 379: 328-331, 2004). Most protein arrays currently available have been observed using enzymes or fluorescence tags (Phelan and Nock, Proteomics , 3: 2123-2134, 2003), but SPR imaging is capable of observing multiple biomolecular interactions on gold thin films. A label-free technique (Rich and Myszka, Biotechnol . , 11: 54-61, 2000).

Protein microarrays or protein chips consist of large amounts of capture agents such as antibodies, proteins or peptides. The protein chip also includes a chip containing an alternative capture agent such as a nucleic acid.

In general, microarrays of single-stranded DNA (ssDNA) have been used primarily for quantitative monitoring of gene expression along with complementary DNA (Schena et. al ., Science, 270: 467-470, 1995).

However, most DNA-binding proteins have been reported to attach to double-stranded DNA (dsDNA) (Bulyk et al. al ., Biotechnol ., 17: 573-577, 1999), the interaction of multiple proteins with DNA-patterned chip surfaces has been analyzed using SPR or SPR imaging systems (Hao et. al ., FEBS Lett . 536: 151-156, 2003; Schaufler et al ., Biol. 329: 931-939, 2003). As an example, the analysis of the correlation between p53 protein and cognate DNA sequences (Maillart et al ., Oncogene , 23: 5543-5550, 2004) and binding of cMyb DNA-binding domains to sequence specific cis-acting elements (transcriptional regulatory sites) (Oda et al. al ., FEBS Lett ., 454: 288-292, 1999).

As a conventional protein immobilization method, a method of immobilizing a protein by integrating an active group (chemical functional group for immobilizing a protein by chemical bonding) on a substrate using plasma (Korean Patent No. 448880), and a sol-gel on a solid substrate surface (sol-gel) method to form a porous sol-gel thin film having a sufficiently increased specific surface area, and then to fix the protein by physical adsorption on the porous thin film (Korean Patent No. 577694), poly by plasma reaction Provided is a method for immobilizing an antithrombogenic protein on a tetrafluoroethylene (PTFE) surface (Korean Patent No. 491700) and a protein immobilizing enzyme for binding an enzyme in which two or more consecutive fusions of cationic amino residues are fused to two enzymes. The method of fixing a protein (Korean Patent Publication No. 10-2003-0034136) and the like have been known.

However, these conventional methods have disadvantages such as denaturation of proteins by chemical processes, deterioration of protein immobilization ability due to protein fixation methods that rely only on physical adsorption, increase of surface modification costs and difficulty in mass production and mass production.

In addition, a method of fixing a protein to a hydrophobic polymer layer bound to a solid support using a substrate (WO 2003/072752), a method of fixing a protein using a buffer component on the plastic surface (EP 0916949) and a hydrophobic in alcohol solution Although a method of fixing a protein by contacting the protein on a solid surface having a surface (WO 2005/070968) has been published, there has not been developed an example of directly fixing a protein at a corresponding site using cognate DNA.

Accordingly, the present inventors have made diligent efforts to solve the problems of the prior art. After modifying the surface of the substrate using cognate DNA of the protein to be immobilized on the substrate, the protein is immobilized on the surface-modified substrate. The present invention was completed by confirming the specific binding to the cognate DNA.

An object of the present invention is to provide a method for selectively immobilizing a protein or peptide on a substrate and a biochip prepared by the method.

In order to achieve the above object, the present invention comprises the steps of: (a) spotting the cognate DNA of the protein or peptide on the substrate surface; And (b) immobilizing the substrate onto which the cognate DNA has been spotted on the surface of the protein or peptide solution to immobilize the protein or peptide.

The present invention also provides a biochip characterized in that the protein or peptide is bound to the cognate DNA spotted on the substrate prepared by the above method.

According to the present invention, a protein to be immobilized on a substrate can be selectively and directly immobilized at a desired position, and a separate process for controlling the orientation of the protein is not required, and thus the protein immobilization method according to the present invention in a biochip and a biosensor If applied, it can expect a simple and economic effect.

As used herein, the term 'site-directed immobilization' is a term used to immobilize a protein on a substrate, and means that the protein is directly immobilized to a specific site without any other means.

As used herein, the term 'cognate DNA' refers to a DNA sequence to which a protein binds.

The present invention comprises the steps of: (a) spotting a cognate DNA which is a DNA sequence that binds to a protein or peptide on the substrate surface; And (b) immobilizing the protein or peptide onto the substrate by immersing the substrate spotted on the surface of the cognate DNA of step (a) in the protein or peptide solution. It is about.

At this time, the protein or peptide solution is prepared using 50 mM phosphate buffer (50 mM phosphate, 0.5 N NaCl, pH 7.4) as a solvent, the concentration of the protein or peptide is preferably 5 ~ 15 μM.

In the present invention, the substrate may be selected from the group consisting of gold, glass, silicon, gold nanoparticles, magnetic nanoparticles and quantum dot nanoparticles, but is not limited thereto.

In the present invention, the protein or peptide may be characterized in that the DNA binding domain is fused.

That is, the protein or peptide may be a fluorescent protein in which a DNA binding domain is fused, an antibody in which a DNA binding domain is fused, an enzyme in which a DNA binding domain is fused, a peptide in which a DNA binding domain is fused, and a DNA binding protein. However, it is not limited thereto.

In the present invention, the cognate DNA may be a DNA including a promoter or a UAS (upstream activating sequence) of a transcriptional regulator, but is not limited thereto.

Herein, the promoter of the transcriptional regulator is a concept of a binding site that binds to a protein. In the present invention, the promoter of the transcriptional regulator is used as a comprehensive meaning including an operator of a transcriptional regulator, and the UAS is a DNA. The concept of DNA recognition of a protein or peptide that binds to it.

GAL4 is one of the yeast galactose metabolic regulators, and two regulators that regulate yeast galactose metabolism are GAL4 and GAL80. In the presence of galactose, the GAL4 protein binds to GAL4-responsive elements that are located above about 20 genes involved in galactose metabolism. In the absence of galactose, the GAL80 protein binds to the GAL4 protein and interferes with the transcriptional activity of the GAL4 protein. . GAL4-responsive elements contain one or more conservative palindromic sequences to which GAL4 proteins bind. This sequence is an upstream activating sequence (UAS) sequence consisting of 17-mers. The higher the copy number of the 17-mer UAS (x1, x2, x3, etc.), the higher the transcriptional activity.

LexA protein acts as a transcription inhibitor of the SOS gene (DNA damage repair gene) of E. coli E. coli . When DNA is damaged, RecA protein activates protease, activated protease decomposes LexA protein, preventing LexA protein from inhibiting transcription of SOS genes, thereby repairing damaged DNA. the repair system is activated. The site where the LexA transcription inhibitor binds to the promoter of the SOS genes is the LexA op (LexA operator) sequence.

In the present invention, the protein is GAL4 DNA-BD: EGFP (Enhanced Green Fluorescent Protein) or LexA DNA-BD: RFP (LexA DNA-Binding Domain: Red Fluorescence Protein), and the cognate DNA is It may be characterized as a UAS or LexA operator gene (LexA operator, LexA op) of GAL4. In addition, the protein is a lambda repressor protein (lambda repressor protein, lambda cI) of the lambda bacteriophage, the cognate DNA may be a lambda operator (lambda operator, lambda operator), but is not limited thereto.

That is, as the protein to be immobilized in the present invention, GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP and cognate DNA of the protein may be used as UAS and LexA op of GAL4, respectively, but are not limited thereto. ).

In the present invention, before the step (a) it may be characterized in that it further comprises the step of forming a poly (L-lysine) monolayer on the surface of the substrate.

The present invention shows that stable and sustained DNA immobilization can be achieved through a polycationic compound bound to the negatively charged phosphate group of dsDNA (Serap et. al . , Polymer International , 52: 169: 1174, 2003). Therefore, it is preferable to use poly (L-lysine), which is a small positively charged synthetic peptide, in the DNA immobilization of the present invention, but is not limited thereto.

In another aspect, the present invention relates to a biochip characterized in that a protein is bound to cognate DNA spotted on a substrate prepared by the above method.

As it is determined that a protein whose orientation has been regulated has both the stability and functionality of the protein, selective immobilization of the protein on the surface of the chip is an important procedure. To date, a variety of methods have been developed for use in protein immobilization in biosensors, and thus, protein immobilization on the chip surface is important for the development of sensitive biosensors (Oh et. al . , FASEB J., 19: 1335-1337, 2005; Seong and Choi, Proteomics, 3: 2176-2189, 2003).

In terms of biomolecule-selective affinity, the DNA-binding domains obtained from two different transcription factors—the yeast GAL4 transcriptase and the E. coli LexA transcription inhibitor—GAL4 DNA-BD having an upstream activating sequence (UAS). Keegan et al . , Science 231; 699-704, 1986; Johnston et al . , Proc. Natl. Acad. Sci., 83: 6553-6557, 1986) and was selected as a fusion tag based on the specific interaction of LexA DNA-BD with LexA operator (Keegan et al. al ., Science 231; 699-704, 1986; Johnston et al ., Proc . Natl . Acad . Sci . USA 83: 6553-6557, 1986), thereby generating GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP fusion proteins.

According to the present invention, by using the principle of selectively binding a cognate DNA with a protein or peptide, the protein or peptide can be oriented and fixed to the substrate selectively and directly.

In addition, in the case of using a fluorescent protein to which DNA BD is bound as a protein to be immobilized, there is an advantage that multiple analysis can be performed by SPR imaging or fluorescence measurement of DNA DNA affinity.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example  1: Protein Immobilization on Gold Chips

1-1: Gene Replication, Expression and Purification of Recombinant Fusion Proteins

In order to replicate the GAL4 DNA-BD: EGFP fusion protein, a partial gene encoding the yeast GAL4 DNA-BD was prepared using a primer (SEQ ID NO: 1: GGA ATC CAT ATG ATG AAG CTA CTG TCT TCT ATC) and a primer (SEQ ID NO: No. 2: GGA ATT CCG GCG ATA CAG TCA ACT G) and amplified by PCR. The DNA fragment was then inserted into the pET-21a: EGFP plasmid using the NdeI / HindIII site to replicate the GAL4 DNA-BD: EGFP fusion protein.

For replication of the LexA DNA-BD: RFP fusion protein, a partial gene encoding E. coli LexA DNA-BD was used as a primer (SEQ ID NO: 3: GGA ATC CAT ATG ATG AAA GCG TTA ACG GCC) and primer (SEQ ID NO: 4: CCC AAG). Amplification by PCR using CTT CAG CCA GTC GCC GTT GCG). Thereafter, the NDEI / HindIII site was used to clone the pET-21a: RFP vector to replicate the LexA DNA-BD: RFP fusion protein.

To purify the cloned GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP fusion proteins, 10 ml of crude cell lysate was placed on an IDA-miniexcellose affinity column (Bioprogen Co., Korea). . The fusion protein was purified by eluting with 5 mL of 0.5 M imidazole in buffer (50 mM phosphate, 0.5 N NaCl, pH 8.0).

1-2: surface treatment of gold thin film and poly (L- lysine ) Monolayer ( monolayer ) formation

The sample 10 (3-mercaptopropyl) trimethoxylane ( Sigma Aldrich, USA) in the back of silanized (silanized) on the cover slide of a microscope (18 x 18 mm ²⁾ gold thin film (47nm) prepared the samples was vapor deposited, Surface treatment was performed by immersing in ethanol MUA (ethanolic mercaptoundecanoic acid) for at least 24 hours.

The sample soaked in MUA was exposed to 75 mM EDC (1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride) and 15 mM NHS (N-hydroxylsuccinimide) in DW (distilled water) for 1 hour. A MUA-NHS ester monolayer was formed. The MUA-NHS ester monolayer and 1 mg / mL poly (L-lysine) (5 mM lysine residue concentration) solution were reacted for 1 to 2 hours in DW, followed by 1 M ethanolamine (pH 8.5) solution. Was soaked for at least 15 minutes to form a poly (L-lysine) monolayer.

1-3: Gold Chip  On dsDNA Microarray ( microarray ) Production and SPR  Imaging measurement

Four different oligonucleotides (UAS _x1 , UAS _x2 , LexA op _x1 and LexA op _x2) were used for the preparation of the dsDNA array on the gold chip, and the sequence of the oligonucleotides is shown in Table 1. In addition, DW (distillated water) was used as a control.

Oligonucleotide order SEQ ID NO: USA _x1 5'-CGGAGGACAGTCCTCCG-3 ' SEQ ID NO: 5 USA _x2 5'-CGGAGGACAGTCCTCCGCGGAGGACAGTCCTCCG-3 ' SEQ ID NO: 6 LexA op _x1 5'-TACTGTATGAGCATACAGTA-3 ' SEQ ID NO: 7 LexA op _x2 5'-TACTGTATGAGCATACAGTATACTGTATGAGCATACAGTA-3 ' SEQ ID NO: 8

A dsDNA was spotted on a gold chip on which a poly (L-lysine) monomolecular layer prepared in Example 1-2 was formed to form a dsDNA microarray. At this time, the diameter of each spot was 330 µm, and the distance between the spots was 350 µm.

The gold chip formed with the microarray was stored at 80% humidity for 30 minutes, and then, in order to remove unbound dsDNA, the gold chip in 3xSSC buffer (150 mM NaCl, 15 mM sodium citrate) containing 0.2% SDS. Was washed three times and then blocked with BSA (5 μl, 1 mg / ml) in 50 mM PBS buffer (pH 7.4) at 25 ° C. for 1 hour.

The gold chip was then immersed in a solution of GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP fusion protein (10 mg / ml) in 50 mM PBS buffer (pH 7.4) at 25 ° C. for 1 hour. After washing with PBS buffer and distilled water, DNA-protein interactions were analyzed using SDS-PAGE gel assay and SPR imaging system (Jung et al ., Biochem . 330: 251-256, 2004).

As a result of 10% SDS-PAGE gel analysis and Coomassie staining, as shown in FIG. 2A, protein markers bound on the gold chip (Lane 1), GAL4 DNA-BD: EGFP is a gold chip The binding phase (Lane 2) and LexA DNA-BD: RFP binding on the gold chip (Lane 3) was confirmed.

In addition, as a result of analyzing the immobilization of GAL4 DNA-BD: EGFP using the SPR imaging system, as shown in FIG. 2B, GAL4 DNA-BD: EGFP binds to UAS _x1 and UAS _x2 , but binds to UAS _x2. From brighter images, it was confirmed that GAL4 DNA-BD: EGFP binds more specifically to UAS _x2 .

In addition, analysis of the immobilization of LexA DNA-BD: RFP using the SPR imaging system, as shown in Figure 2C, LexA DNA-BD: RFP binds to LexA op _x1 and LexA op _x2 , LexA op Brighter images were confirmed by binding to _x2 , indicating that LexA DNA-BD: RFP binds more specifically to LexA op _x2 .

On the other hand, when using the DW as a control, no image was confirmed using the SPR imaging system.

1-4: Kin DNA Effect of Concentration on Protein Immobilization

In the protein immobilization according to the present invention, to confirm the effect of the cognate DNA concentration on the protein, 0.5 ~ 100.0 pM UAS _{× 2} and LexA op _{× 2} spotted on a gold chip, respectively, 10 μM GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP proteins.

As a result, as shown in FIG. 3A, the relative SPR imaging signal intensity of GAL4 DNA-BD: EGFP binding was found to be proportional to the concentration of cognate DNA UAS _{× 2} . Further, as shown in Figure 3 B, similarly, LexA LexA DNA-BD for op _{× 2:} SPR imaging relative signal strength of the binding of RFP is shown to be proportional to the concentration of the LexA op _{× 2} of homologous DNA.

1-5: Effect of Protein Concentration on Immobilization of the Protein

In the protein immobilization according to the present invention, to confirm the effect of the concentration of the protein, 50 pM UAS _x2 And 1.0 μM, 2.0 μM, 4.0 μM, 7.0 μM, 10.0 μM, and 13.0 μM of GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP, respectively, on gold chips spotted with 25 pM LexA op _{× 2} The SPR imaging signal intensity was then examined.

As a result, as shown in Fig. 4A and 4B, UAS _x2 And the binding strength of GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP to 25 pM LexA op _{× 2} was shown to be proportional to the concentration of the GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP.

Example  2: From glass chips DNA Cognates of Binding Proteins dsDNA Fluorescence of specific binding to

To observe site-direct immobilization using fluorescence, glass chips (15 × 9 mm) were coated with poly (L-lysine) and then spotted with a 114 dsDNA array consisting of UAS _{× 2} and LexA op _{× 2} It was. Here, 66 spots were arrayed at UAS _{× 2} , 48 spots were spotted at LexA op _{× 2} , and then labeled 'DNA'.

Thereafter, purified 10 μM GAL4 DNA-BD: EGFP and 50 pM LexA DNA-BD: RFP protein mixture were applied to the glass chip surface modified with dsDNA consisting of 114 spots. At this time, the GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP are fusion proteins fused with EGFP and RFP at the C-terminus of each protein for fluorescence observation.

As a result, as shown in FIG. 5, the green and red fluorescence images were separated and observed. 66 green fluorescence images were formed as a result of site-direct immobilization of GAL4 DNA-BD: EGFP protein to UAS _{× 2} , a GAL4 DNA-BD-specific binding site. In contrast, the 48 red fluorescent spots labeled 'DNA' were formed by site-direct immobilization between LexA DNA-BD-specific binding sites, LexA op _{× 2} and LexA DNA-BD: RFP protein.

Thus, specific binding of GAL4 DNA-BD: EGFP and LexA DNA-BD: RFP proteins to UAS _{× 2} and LexA op _{× 2,} respectively, was verified. Observations based on fluorescence-based detection were found to be consistent with site-specific immobilization of the recombinant DNA-binding protein for the corresponding DNA element, as monitored by the SPR imaging system.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 shows that GAL4 DNA-BD: EGFP fusion protein and LexA DNA-BD: RFP fusion protein are selectively immobilized on the surface of the gold chip surface modified with dsDNA consisting of UAS and LexA op of GAL4, their cognate DNA, respectively. And a schematic representation of monitoring the immobilized results using an SPR image system.

2 shows that GAL4 DNA-BD: EGFP fusion protein and LexA DNA-BD: RFP fusion protein are selectively immobilized on the surface of the gold chip surface modified with dsDNA consisting of UAS and LexA op of GAL4, each of their cognate DNA. (A) shows the results of the SDS-PAGE gel assay, (B) shows the results of the analysis of the immobilization of the GAL4 DNA-BD: EGFP fusion protein using the SPR image analysis, (C ) Shows the results of analysis of immobilization of LexA DNA-BD: RFP fusion protein using SPR image analysis.

Figure 3 shows that when GAL4 DNA-BD: EGFP fusion protein and LexA DNA-BD: RFP fusion protein are selectively immobilized on the surface of the gold chip surface modified with dsDNA consisting of UAS and LexA op of GAL4, their respective cognate DNA, respectively. In addition, the concentrations of UAS and LexA op of GAL4 are shown in the immobilization of the fusion protein.

Figure 4 shows that when GAL4 DNA-BD: EGFP fusion protein and LexA DNA-BD: RFP fusion protein are selectively immobilized on the surface of the gold chip surface modified with dsDNA consisting of UAS and LexA op, each of their cognate DNAs, The effect of concentrations of GAL4 DNA-BD: EGFP fusion protein and LexA DNA-BD: RFP fusion protein on immobilization is shown.

FIG. 5 shows GAL4 DNA-BD: EGFP fusion protein and LexA DNA-BD: RFP fusion protein selectively immobilized on a glass slide surface modified with dsDNA consisting of UAS and LexA op of GAL4, their cognate DNA, respectively. The results shown in fluorescent colors are shown.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for Immobilizing Proteins on a Cognate DNA-Modified          Substrate Surface <130> P08-B093 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggaatccata tgatgaagct actgtcttct atc 33 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggaattccgg cgatacagtc aactg 25 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggaatccata tgatgaaagc gttaacggcc 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cccaagcttc agccagtcgc cgttgcg 27 <210> 5 <211> 17 <212> DNA <213> yeast GAL4 Cognate DNA <400> 5 cggaggacag tcctccg 17 <210> 6 <211> 34 <212> DNA <213> yeast GAL4 Cognate DNA <400> 6 cggaggacag tcctccgcgg aggacagtcc tccg 34 <210> 7 <211> 20 <212> DNA <213> E. coli LexA Cognate DNA <400> 7 tactgtatga gcatacagta 20 <210> 8 <211> 40 <212> DNA <213> E. coli LexA Cognate DNA <400> 8 tactgtatga gcatacagta tactgtatga gcatacagta 40  

Claims (7)

A method of selectively immobilizing a protein or peptide on a substrate, comprising the following steps: (a) spotting cognate DNA of a protein or peptide on a substrate surface; And (b) immobilizing the substrate onto which the cognate DNA is spotted on the surface of the protein or peptide solution to immobilize the protein or peptide. The method of claim 1, wherein the substrate is selected from the group consisting of gold, glass, silicon, gold nanoparticles, magnetic nanoparticles, and quantum dot nanoparticles. The method of claim 1, wherein the protein or peptide is characterized in that the DNA binding domain is fused. The method of claim 1, wherein the cognate DNA is a DNA comprising a promoter or upstream activating sequence (UAS) of a transcriptional regulator. The method of claim 1, wherein the protein is GAL4 DNA-BD: EGFP (Enhanced Green Fluorescent Protein) or LexA DNA-BD: RFP (LexA DNA-Binding Domain: Red Fluorescence Protein). Is UAS or LexA op (operator) of GAL4. The method of claim 1, further comprising forming a poly (L-lysine) monolayer on the surface of the substrate before step (a). Biochip, characterized in that the protein or peptide is bound to the cognate DNA spotted on the substrate prepared by the method of any one of claims 1 to 6.

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