WO2005108609A1

WO2005108609A1 - Method for identification and analysis of certain molecules using the dual function of single strand nucleic acid

Info

Publication number: WO2005108609A1
Application number: PCT/KR2005/001294
Authority: WO
Inventors: Sung Chun Kim
Original assignee: GENOPROT Inc
Current assignee: GENOPROT Inc
Priority date: 2004-05-06
Filing date: 2005-05-04
Publication date: 2005-11-17
Anticipated expiration: 2006-11-06

Abstract

The present invention relates to a method for the identification and quantitative analysis of specific substances. More particularly, the present invention provides a very useful method for identifying specific substances and investigating and analyzing the quantitative change of the specific substances using the dual function of single-stranded nucleic acids which form complexes with the specific substances or form double-stranded nucleic acids with com¬ plementary single-stranded nucleic acids.

Description

Description METHOD FOR IDENTIFICATION AND ANALYSIS OF CERTAIN MOLECULES USING THE DUAL FUNCTION OF SINGLE STRAND NUCLEIC ACID Technical Field

[1] The present invention relates to a method for the identification and quantitative analysis of specific substances, and more particularly, to a new and simple method for identifying specific substances and investigating and analyzing the quantitative change of the specific substances, using the dual function of single-stranded nucleic acid aptamers which form complexes with the specific substances or form double-stranded nucleic acids with complementary single-stranded nucleic acids.

[2] Background Art

[3] Although many technologies for the identification and the investigation and analysis of quantitative change of specific substances have been developed with the development of physics and biochemistry, there is a high need for a new and efficient method due to problems on the use, maintenance cost, easiness, sensitivity, test time and process automation of the existing methods or devices. A method for analyzing specific substances is a part of the entire process, but not an object to itself, and this method can be useful for the identification of microorganisms and viruses, the analysis of cells, proteins and organic substances, and the like, so is widely applied in medicine, veterinary, environmental engineering, food engineering, agricultural industry, and the like.

[4] Nucleic acid is a polymer of covalently linked nucleotides which are small organic compounds, including phosphate, deoxyribose, and purine (adenine or guanine) or pyrimidine (cytosine, thymidine or uracil). Nucleic acid exists as single strands or double strands, in which the single strands bind to each other by the hydrogen bonding or interaction between nucleotides to form an unique stereostructure which is determined by a single-stranded base sequence. Nucleic acids, such as deoxyri- bonucleic acid (DNA) and ribonucleic acid (RNA), are generally reservoirs of information for the expression of proteins with cellular structures and activities, such as enzymatic activity. However, since it was reported in 1982 that RNA has enzymatic activity by forming a specific structure, there have been many reports on the structural characteristics and specific functions of nucleic acids. Nucleic acid consists of repeating units of four bases and maintains high diversity to form many sterostructures which are stabilized by forming complexes by interaction with specific substances. [5] Nucleic acid can act as one ligand for specific substances including proteins. From a library where single-stranded nucleic acids with various base sequences, nucleic acids with high binding affinity and specificity to specific substances are selected by a given selection process and base sequencing. A technique for selecting nucleic acids binding to specific substances is called "SELEX" (Systematic Evolution of Ligand by Exponential enrichment), and the selected products, nucleic acids, are called "aptamers" (Tuerk C. and Gold L.; Science, 249, pp505-510, 1990). By SELEX, molecules are selected which bind with high affinity to not only proteins capable of binding to nucleic acids in a natural condition but also various biomolecules, including proteins not bound to nucleic acids. Technologies for identifying and analyzing specific substances on the basis of the single-stranded nucleic acids thus selected are developed using a molecular beacon (Li JJ et al.; Biochem. Biophys. Res. Commun., 292m. pp31-40, 2002), a calorimetric method (Stojanovic MN, Landry DW.; J. AM. CHEM. SOC, 124. pp9678-9679, 2002), an electrochemiluminescence and enzymatic method (Bruno JG, Kiel JL.; Biotechniques; 32(1), ppl78-80, ppl82-3, 2002) and the like, however, the technology for identifying and analyzing specific substances in an array manner is not yet specifically reported.

[6] The identification and analysis of specific substances are frequently performed using physical and physical properties, and an analysis with the use of the specificity and affinity between substances is generally conducted by methods developed on the basis of immunological methods. The immonological methods are techniques in which an antibody binds to a specific antigen contained in a sample by antigen-antibody reaction to form a complex which is then analyzed by a labeled secondary antibody. For example, ELIS A is a method for analyzing a specific antigen by allowing a primary antibody fixed to a solid medium to react with a sample containing an antigen and treating the resulting complex with a labeled secondary antibody recognizing the complex. As the labeled secondary antibody recognizes and binds to the primary antibody-antigen complex fixed to the solid medium, the marker binds to the solid medium. Probes used in ELISA are labeled with fluorescent indicators, dioxygenin, horseradish peroxidase, alkaline phosphatase and the like.

[7] Recently, protein chips are developed and used to analyze a large amount of proteins in high speed. The protein chips allow the identification and the investigation and analysis of quantitative amount of specific substances since the amino acid sequence of an antibody is known. Methods for fabricating the protein chips include a method comprising spotting antibodies by Microarrayer. A protein chip detection technology must capable of detecting very weak signals generated in a state where various antibodies are integrated in a narrow area at a high density in order for one protein chip to provide as much information as possible. Also with an increase in bio- information on proteins, the degree of integration of the proteins gradually increases, and thus, a new method for quantitatively the proteins in a rapid and precise manner is needed. For detection, laser-induced fluorescence has mainly been used up to date, and electrochemical detection methods and the like are now developed. As described above, although the methods for the identification of specific proteins and the investigation and analysis of quantitative amount of the proteins with the use of protein chips by various processes have been developed, but have problems in that expensive devices and reagents are used and complicated processes must be conducted.

[8] Accordingly, the present inventors prepared a specific substance-target probe complex by immobilizing capture probes onto a solid substrate to fabricate an array and treating the array with an analytical reagent, and then, performed a method for identifying and quantifying specific substances using a single-stranded nucleic acid array, comprising the steps of adding a hybridization solution to the specific substance- target complex to dissociate the target probes, reacting the capture probes with labeled target probes, and investigating substances labeled to the target probes by laser- induced fluorescence, and performed a method for identifying specific substances and analyzing the quantitative change of the specific substances, comprising the steps of reacting single-stranded nucleic acids with a sample, washing the aptamer-specific substance complexes, dissociating the single-stranded nucleic acids from the specific substance-aptamer complexes, and investigating the dissociated nucleic acids by a nucleic acid analysis technique, thereby completing the present invention.

[9] Disclosure of Invention Technical Problem

[10] It is an object of the present invention to provide a system for investigating and analyzing various biomolecules, including microorganisms, cells and proteins, by identifying specific substances and analyzing the quantitative change of the substances using the dual function of single-stranded nucleic acid aptamers which form complexes with specific substances in a given environment or form double-stranded nucleic acids with complementary single-stranded nucleic acids.

[11] Technical Solution

[12] To achieve the above object, the present invention provides a method for identifying and analyzing specific substances using a single-stranded nucleic acid array, the method comprising the steps: (1) immobilizing single-stranded nucleic acid capture probes each consisting of reactive group (X)-spacer site (R)-target probe recognition site (V) on a solid substrate so as to fabricate an array; (2) reacting specific substances with target probes by treatment with an analytical reagent so as to prepare specific substance-target probe complexes; (3) washing and isolating the specific substance-target probe complexes prepared in the step (2); (4) dissociating the target probes from the isolated complexes or amplifying and labeling the target probes; (5) reacting the capture probes with labeled target probes; and (6) investigating substances labeled to the target probes.

[13] The specific substance may be at least one selected from the group consisting of bacteria, fungi, virus, cell lines, tissues, proteins isolated therefrom, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, and enzymes.

[14] In another aspect, the present invention provides an analysis kit for the identification and quantitative analysis of E. coli, comprising: an array fabricated with single-stranded nucleic acid capture probes having base sequences selectively binding to E. coli; an analytical reagent containing single-stranded nucleic acid or 0.2% BSA, which is used in the binding reaction between target probes and E. coli; a hybridization solution; a 5'-primer labeled with Cy-5 and a 3'-primer.

[15] Hereinafter, the present invention will be described in detail.

[16] In one aspect, the present invention provides a method for identifying and analyzing specific substances using a single-stranded nucleic acid array, the method comprising the steps: (1) immobilizing single-stranded nucleic acid capture probes each consisting of reactive group (X)-spacer site (R)-target probe recognition site (N) on a solid substrate so as to fabricate an array; (2) reacting specific substances with target probes by treatment with an analytical reagent so as to prepare specific substance-target probe complexes; (3) washing and isolating the specific substance-target probe complexes prepared in the step (2); (4) dissociating the target probes from the isolated complexes or amplifying and labeling the target probes; (5) reacting the capture probes with labeled target probes; and (6) investigating substances labeled on the target probes. The present invention is a method for the identification and quantitative analysis of specific substances by the use of the dual function of single-stranded nucleic acid aptamers which, as shown in FIG. 1, form complexes with specific substances in SELEX selection buffer or form double-stranded nucleic acids with complementary single-stranded nucleic acids in a hybridization solution.

[17] The step (1) of the inventive method is the step of fabricating an array having si ngle-stranded nucleic acid capture probes attached thereto. Specifically, the present invention provides single-stranded nucleic acids as capture probes to be immobilized on a single-stranded nucleic acid array, in which the single-stranded nucleic acids each consisting of three portions, i.e., reaction group (X)-spacer site (R)-target probe recognition site (V), are constructed. [18] The capture probes are elements having a very great effect on the extent of hybridization, and the determination of their base sequences is a very important step. Each of the probes forming the array consists of characteristic bases, and their complexes must be maintained at suitable Tm. Accordingly, the hybridization degree of the complex probes should be so that the complex probes can maintain their own signal values without contamination with other fluorescence-labeled target probes.

[19] The target probe recognition site (N) is based on the base sequence of a single- stranded nucleic acid selected by SELEX. Preferably, it is a single-stranded target probe which is complementary to the target probe containing a recognition site for a specific substance and has a base sequence of 50-100 bp. The target probe recognition site contains the base sequence of a single-stranded nucleic acid of SEQ ID ΝOS: 3-26 specifically binding to E. coli, which was isolated and analyzed by the present inventors (Korean Patent Application No. 2002-0064027), among single-stranded nucleic aptamers selected by the standard SELEX method (Bock LC et al., Nature, 335(6360), pp564-6, 1992) in the present invention.

[20] The base sequence of the spacer site (R) may be designed for each of specific substances to be fundamentally identified or may be used for the specific substances in common. Points to be considered in the actual design of the spacer include the sequence length, the base composition, the presence or absence of a non- complementary base sequence, and self-complementarity. The spacer site is considered in view of problems occurring in attaching the capture probes to the substrate surface, and examples thereof include single-stranded nucleic acids having a base sequence of 10-30 bp and peptides having an amino acid sequence of 5-20 amino acids. In a preferred embodiment of the present invention, a base sequence of SEQ ID NO: 1 is provided.

[21] Also, the reactive group (X) site is a site bonded with reactive groups attaching the single-stranded nucleic acids to the substrate of the array, and as the reactive groups, amine groups, thiol (-SH) groups, biotin and the like may be used.

[22] The single-stranded nucleic acid capture probes according to the present invention are preferably single-stranded nucleic acids each consisting of a 5'-primer (SEQ ID NO: 1) modified with an amine group at the 5 '-end, each of base sequences of single- stranded nucleic acids of SEQ ID NOS: 3-26, and a 3'-primer (SEQ ID NO: 2). These capture probes may be prepared by isolating single-stranded nucleic acids specifically binding to E. coli, among single-stranded nucleic acids selected by the standard SELEX method, and then repeatedly subjecting the isolated nucleic acids to PCR with primers of SEQ ID NOS: 1 and 2.

[23] Moreover, substrates which can be used in the fabrication of the single-stranded nucleic acid array according to the present invention include those made of an inorganic material, such as glass or silicon, acryl resin, or a polymer material, such as PET (polyethylene terephtalate), polycarbonate, polystyrene, or polypropylene. Preferred is a slide glass. The substrate used is coated with aldehyde, poly L- lysine or the like.

[24] More specifically, the capture probes to be fixed on the glass slide are prepared by performing PCR using plasmid templates having cloned single-stranded nucleic acids selected by the standard SELEX method and using 3 '-primer and 5 '-primer modified with an amine group at the 5 '-end. Unbound primers are removed by a PCR product purification kit, and nucleic acid capture probes to be immobilized to the slide are prepared. Using an aldehyde-coated slide as the glass slide, an array having single- stranded nucleic acids arranged thereon as shown in FIG. 6 is prepared. The fabrication of the array is performed using a pin-type Microarrayer system (GenPak) in such a manner that the spot center-center spacing is 350-400 mm. Each of the single-stranded nucleic acids is dissolved in 3xSSC buffer to adjust the concentration, and the humidity within the arrayer is maintained at 70-80% to perform the spotting. The spotted slides are left to stand in a humidified chamber and baked at 80 °C for 2-4 hours. After the single-stranded nucleic acids are immobilized on the glass slide by the method known in the art, the slide is dried by centrifugation and then stored in a light-shielded condition before use.

[25] The array having the single-stranded nucleic acids arranged thereon (FIG. 1) can be fabricated in various methods known in the prior art (M. schena; DNA microarray; a practical approach, Oxford, 1999), and the configuration of the array having the single- stranded nucleic acids attached thereon is shown in FIG. 2.

[26] The step (2) of the present invention is the step of reacting specific substances with target probes by treatment with an analytic reagent so as to prepare specific substance- target probe complexes. The target probes are prepared by either labeling single- stranded nucleic acid aptamers binding to specific substances or labeling single- stranded nucleic acids complementary to the base sequences of the single-stranded nucleic acid aptamers, in which various markers capable of recognizing signals smoothly in the investigation step can be used. In the former case, the target probes are single-stranded nucleic acids having a structure capable of specifically binding to either the single-stranded nucleic acids selected by SELEX or target substances. For this purpose, the secondary structure of structural free energy of the nucleic acids are examined using the MFOLD program modeling the secondary structure of nucleic acids, and then, single-stranded nucleic acids having the most stable secondary structure are selected.

[27] Examples of a marker of the target probes, which can be used in the present invention, include a radioactive isotope, a fluorescent substance, such as fluorescein, Cy3 or Cy5, and biotin. The fluorescent substance is preferably used. The specific substance is at least one selected from the group consisting of bacteria, fungi, virus, cell lines, tissues, proteins isolated therefrom, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies and enzymes. The cell lines and the tissues include those derived from eukaryotes or prokaryotes. The eukaryotes indicate human beings, animals and plants.

[28] With respect to the concrete content of the reaction step, in the case where the target probes are prepared by labeling single-stranded nucleic acid aptamers binding to the specfic substances, the analysis method is shown in FIG. 4, and in the case where the target probes are prepared by labeling single-stranded nucleic acid complementary to the single-stranded nucleic acid apamers, the analysis method is shown in FIG. 5. The former case was named an "aptamer probe method", and the latter case was named an "antiaptamer probe method".

[29] The aptamer probe method is based on a reaction between the specific substances and the target probes (labeled aptamers), and the antiaptamer probe method is a reaction between the specific substances and unlabeled aptamers, in which the reaction solution comprises a salt composition allowing the single-stranded nucleic acids to bind well to the specific substances, and an element preventing non-specific binding, and the like.

[30] The analytical reagent contains: (1) 20-100 mM, and preferably 50 mM Tris-HCl buffer (pH 7.4); (2) 0-200 mM of each of potassium chloride, sodium chloride and magnesium chloride, and preferably 5 mM potassium chloride, 100 mM sodium chloride, and 1 mM magnesium chloride, which are substances involved in the stabilization of secondary structure of the single-stranded nucleic acids; (3) 0.05-0.2% of sodium azide, and preferably 0.1% sodium azide; and (4) 0.1-0.3%, preferably 0.2% of bovine serum albumin for the inhibition of non-specific binding, or nucleic acid for the inhibition of non-specific binding, the nucleic acid being determined in view of the specificity of the probes.

[31] The analytical reagent comprises at least three of the above-mentioned four fundamental components. The reaction is carried out at a lower temperature than that of SELEX, and preferably a temperature of 20-37 °C. Generally, proteins and singl e- stranded nucleic acids are treated with an excess of the analytical reagent in order to prevent the non-specific binding of the target probes. As the target probes, yeast tRNA, salmon sperm DNA or human placental DNA may be used.

[32] The step (3) of the present invention is the step of washing and isolating the prepared specific substance-target probe complexes to remove target probes of low affinity from the specific substance-target probe complexes. The isolation can be performed by membrane filtration, adsorption or centrifugation. If the specific substances used in the step (2) are microorganisms or cells, the target probe solution is mixed and reacted with a selection buffer containing the sample at 37 °C for 30 minutes, and just then, the resulting complexes are washed with SELEX buffer of various concentrations, distilled water or 0.05M EDTA by centrifugation or filtration through a 0.42-mm membrane so as to remove unbound target probes and target probes of low affinity, before the complexes are used in the second reaction. The washing buffer is preferably 0-1 x SELEX buffer or 0-500 mM EDTA solution. However, if the specific substances are proteins, the single-stranded nucleic acid-protein complexes and unbounded single-stranded nucleic acids may be separated by filtration through a 0.45-mm nitrocellulose membrane, or adsorption of the protein sample to an ELIS A plate, or capillary electrophoresis.

[33] The step (4) of the present invention is the step of dissociating the isolated specific substance-target probe complexes into the specific substances and the target probes or amplifying and labeling the complexes.

[34] In the aptamer probe method, the dissociated specific substance-target probe complexes are used in the step (5). However, in the antiaptamer probe method, the supernatant isolated by centrifugation is added with labeled single-stranded nucleic acids complementary to the single-stranded nucleic acid aptamers, and the mixture is allowed to react for 0-30 minutes, and the reaction product is used in the step (5). The reaction in the step (5) is the step of prehybridizing the array having the single- stranded nucleic acid capture probes attached thereto in a hybridization solution, treating the array with the reaction product of the step (4), and washing and drying the resulting array.

[35] The step (5) of the present invention is the step of reacting the capture probes with the labeled target probes. The most important in the step (5) are hybridization rate and the stability of the hybrid. External factors influencing them include temperature, ionic strength, formamide and other polymers, and internal factors include DNA length and sequence complexity, and the environment of DNA in the case of mixed phase hybridization.

[36] In the identification and quantitative analysis of biomolecules by the array having single-stranded nucleic acids attached thereto according to the present invention, a nucleic acid hybridization solution for the specific binding between the single-stranded nucleic acid capture probes and the target probes is provided. The hybridization solution comprises: (1) 0.5-1.5 M sodium chloride, (2) 0-1.0 M sodium citrate, (3) 0.1-0.3% bovine serum albumin for the inhibition of non-specific binding, 0-1.0% SDS, 0-10-fold Denhart solution or 0-1% nonfat dried milk, (4) nucleic acid for the inhibition of non-specific binding, and (5) 30-60 % (v/v) formamide. Preferably, the hybridization solution comprises 1 M sodium chloride, 0.3 M sodium citrate, 0.5% SDS or 100 mg/ml salmon sperm DNA, 0.2% bovine serum albumin or single-stranded nucleic acid. After completion of hybridization, the array is washed with 0.1 IX SSC, 0.2% SDS and the like, at a temperature of 22-70 °C for 30 minutes, and with distilled water for 30 seconds, and then dried in a dark condition. Since the first washing solution is most important in the optimization of washing conditions, the stringency in this step is controlled by the use of various washing solutions. Examples of the washing solution composition include IX SSC + 0.2% SDS, IX SSC + 0.2% SDS, 0.5X SSC + 0.2% SDS, and 0.01X SSC + 0.2% SDS.

[37] The binding extent between the specific substances and the target probes or the binding extent between the target probes and the capture probes is determined depending on the specificity and binding affinity between each other, and from this binding extent, the specific substances can be identified and quantified. The specificity and binding affinity between the single-stranded nucleic acid aptamers and the specific substances change the binding extent of the complexes and determine the kind and amount of the specific substance-single stranded nucleic acid complexes. The target probes (i.e., single-stranded nucleic acids) dissociated from the complexes on the array influence the kind and amount of the target probe-capture probe double strands and determine the fluorescence emitted from the formed double strands. Thus, the analysis of the emitted fluorescence allows the kind and quantitative change of the specific substances.

[38] The step (6) of the present invention is the step of investigating the substances labeled to the target probes by various methods as shown in FIGS. 6 and 7, depending on a marker of the target probes. In the method as shown in FIG. 6, the array 7 is placed on the plate 6, and then, light irradiated from the lamp 11 reaches the spectrometer 9 through the excitation filter 10. The light is irradiated on the array 7 through the lens 8. Fluorescence emitted from the array 7 is collected on the spectrometer 9 through the lens 8, and then, reaches the CCD camera 13 through the emission filter 12. This camera is connected to the CPU 14 so that it can analyze the specific substances by forming images on the NCR 15 and the monitor 16. FIG. 7 shows a measurement method based on ellipsometry which measures the change in polarization state of reflected light. With an increase in the density of spots by the binding between the capture probes and the specific substances in the spots, the polarization state of a plane-polarized light beam reflected from the surface is changed, and this change can be monitored directly in a solution. In a typical method, monochromatic light irradiated from the He-Νe laser 17 is converted into plane- polarized light by the polarizer 18 and irradiated on the surface of the sample and then reaches the detector 21. The compensator 19 converts an elliptically polarized reflected beam into a plane-polarized light. The corresponding angle is measured by the analyzer 29, and ellipsometric parameters, Psi and Delta, are calculated, from the calculated values, the specific substances bound to the capture probes can be identified (Azzam et al, Ellipsometry and Polarized Light, North-Holland Publishing Company: Amsterdam, 1977).

[39] In another aspect, the present invention provides an analysis kit for the identification and quantitative analysis of E. coli, comprising: an single-stranded nucleic acid array comprising capture probes having base sequences specifically binding to E. coli; an analytical reagent containing single-stranded nucleic acid or 0.2% BSA, which is used in the binding reaction between target probes and E. coli; a hybridization solution which is used in the binding reaction between the target probes and the capture probes; a 5 '-primer and a 3 '-primer labeled with Cy-5.

[40] The present invention provides a system for investigating and analyzing biomolecules, including microorganisms, cells and proteins, in a sample, using an array having single-stranded nucleic acids attached thereto.

[41] The system for the investigation and analysis of biomolecules comprises the inventive array having single-stranded nucleic acids immobilized thereon, an analytical reagent, and a means for investigating a marker. Examples of the biomolecules include microorganisms, such as bacteria or virus, cell lines, and proteins and enzymes isolated therefrom.

[42] The array having single-stranded nucleic acid immobilized thereon means an array having the inventive capture probes each consisting of reactive group-spacer site-target probe recognition site, immobilized on a slide glass. The analytical reagent includes a selection buffer, a selection buffer containing 0.2% bovine serum albumin (BSA), or an analytical reagent containing single-stranded nucleic acid. For the investigation of the signaling substances, laser-induced fluorescence or ellipsometric detection may be used, and the obtained data can be analyzed by various bioinformational methods. In a preferred embodiment of the present invention, the laser-induced fluorescence is used. The identification and analysis system can be used to analyze biomolecules.

[43] In still another aspect, the present invention provides a method for identifying and quantifying specific substances using single-stranded nucleic acid probes, the method comprising the steps of: (1) constructing single-stranded nucleic acid probes each consisting of either immobilization site (X)-specific substance recognition site (N)-immobilization site (Y) or fluorescent substance (F)-specific substance recognition site; (2) reacting specific substances with the probes constructed in the step (1) by treatment with an analytical reagent so as to prepare specific substance-probe complexes; (3) washing and isolating the specific substance-probe complex prepared in the step (2); (4) dissociating the probes in the complexes; and (5) investigating the dissociated probes. [44] Examples of the specific substances include bacteria, fungi, virus, cell lines, tissues, proteins isolated therefrom, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, and enzymes.

[45] In still another aspect, the present invention provides an analysis kit for the identification and quantitative analysis of E. coli, comprising: probes based on the base sequences of single-stranded nucleic acid aptamers specifically binding to E. coli; an analytical reagent containing single-stranded nucleic acid or 0.2% BSA, which is used in the binding reaction between the probes and E. coli; a reagent which is used in the isolation of E. coli-probe complexes; and forward and reverse primers.

[46] Hereinafter, the present invention will be described in more detail.

[47] The present invention provides a method for identifying and quantifying specific substances using single-stranded nucleic acid probes, the method comprising the steps of: (1) constructing single-stranded nucleic acid probes each consisting of either immobilization site (X)-specific substance recognition site (V)-immobilization site (Y) or fluorescent substance (F)-specific substance recognition site (N); (2) reacting specific substances with the probes constructed in the step (1) by treatment with an analytical reagent so as to prepare specific substance-probe complexes; (3) washing and isolating the specific substance-probe complexes prepared in the step (2); (4) dissociating the isolated complexes into the probes; and (5) investigating the dissociated probes. As shown in FIG. 11, the present invention is a method for identifying and quantitatively analyzing specific substances, in which the probes are formed into complexes with specific substances in a selection buffer, and then, the probes separated from the formed complexes are analyzed by a nucleic acid analysis technique. This allows the analysis of the sample by the measurement of unbound probes.

[48] The step (1) of the present invention is the step of constructing the single-stranded nucleic acid probes. Specifically, the present invention provides the single-stranded nucleic acid probes each consisting of either immobilization site (X)-specific substance recognition site (V)-immobilization site (Y) or fluorescent substance (F)-specific substance recognition site (N).

[49] The single-stranded nucleic acid probes in the present invention is fabricated in view of the base sequences of the aptamers recognizing the specific substances and the analysis technique in the investigation step. In the case of analysis by a PCR (polymerase chain reaction) technique, single-stranded nucleic acids each consisting of three portions of immobilization site (X)-specific substance recognition site (N)-immobilization site (Y), and in the case of analysis by a fluorescent analysis technique, single-stranded nucleic acids each consisting of two portions of fluorescent site (F)-specific substance recognition site (V).

[50] The specific substance recognition site (N) is based on the base sequences of single- stranded nucleic acids selected by SELEX. Preferably, they are single-stranded target probes which are complementary to the target probes containing a recognition site of specific substances and have a base sequence of 16-60 bp. The specific substance recognition site contains the base sequences of single-stranded nucleic acids of SEQ ID NOS: 3-26 specifically binding to E. coli, which were isolated and analyzed by the present inventors (Korean Patent Application No. 2002-0064027), among single- stranded nucleic aptamers selected by the standard SELEX method (Bock LC et al., Nature, 335(6360). pp564-6, 1992) in the present invention.

[51] If the investigation step is performed by a PCR technique, the immobilization site (X or Y) of the probes has a very great effect on the amplification of the probes, and the base sequencing of the immobilization site is a very important step. The immobilization site of the probes is composed of characteristic bases, and must be maintained a suitable Tm to bind to primers in the PCR step. Thus, the formation of the PCR products should be so that they can maintain their own signal values without contamination. The base sequence of the immobilization site may be designed for each of specific substances to be fundamentally identified or may be used for the specific substances in common. Points to be considered in the actual design of the immobilization site include the sequence length, the base composition, the presence or absence of a non-complementary base sequence, and self-complementarity. In a preferred embodiment of the present invention, the immobilization site (X) has a sequence of SEQ ID NO: 27 (5'-ATA CCA GCT TAT TCA ATT-3', and the immobilization site (Y) has a sequence of SEQ ID NO: 28 (5'-AGA TAG TAA GTG CAA TCT-3').

[52] If the investigation step is performed by fluorometry, a radioactive isotope, a fluorescent substance, such as fluorescein, Cy3 or Cy5, or biotin, may be used as a marker (F) of the probes. The fluorescent substance is preferably used. Examples of the specific substances include bacteria, fungi, virus, cell lines, tissues, proteins isolated therefrom, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, and enzymes. The cell lines and the tissues include those derived from eukaryotes or prokaryotes. The eukaryotes indicate human beings, animals and plants. The specific substances are preferably Escherichia coli. Es- cherichia coli are gram-negative bacilli residing in the intestines of warm-blooded animals and having flagella. These bacilli are generally not pathogenic in the intestines, but often cause cystitis, pyelitis and cholecystitis in the urethra or the biliary tract, and acute enteritis for babies. They are indicative of feces contamination and frequently used in water examination.

[53] The step (2) of the present invention is the step of reacting the specific substance with the probe by treatment with an analytical reagent so as to prepare a specific substance-probe complex. The reaction solution consists of a salt composition allowing the probe to bind well to the specific substance, an element for preventing non-specific binding, and the like.

[54] The analytical reagent contains: (1) 20-100 mM, and preferably 50 mM Tris-HCl buffer (pH 7.4); (2) 0-200 mM of each of potassium chloride, sodium chloride and magnesium chloride, and preferably 5 mM potassium chloride, 100 mM sodium chloride, and 1 mM magnesium chloride, which are substances involved in the stabilization of secondary structure of the single-stranded nucleic acids; (3) 0.05-0.2% of sodium azide, and preferably 0.1% sodium azide; and (4) 0.1-0.3%, preferably 0.2% bovine serum albumin for the inhibition of non-specific binding, or nucleic acid for the inhibition of non-specific binding, the nucleic acid being determined in view of the specificity of the probes. The analytical reagent comprises at least three of the above- mentioned four fundamental components. Preferably, the analytical reagent contains 50 mM Tris-HCl (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide and 0.2% bovine serum albumin. The reaction is carried out at a lower temperature than that in SELEX, and preferably a temperature of 20-37 °C. Generally, proteins and single-stranded nucleic acids are treated with an excess of the analytical reagent in order to prevent the non-specific binding of the target probe. As the target probe, yeast tRNA, salmon sperm DNA or human placental DNA may be used.

[55] The step (3) of the present invention is the step of washing and isolating the prepared specific substance-target probe complex. The isolation can be performed by membrane filtration, adsorption or centrifugation. If the specific substances used in the step (2) are microorganisms or cells, the target probe solution is mixed and reacted with a selection buffer containing the sample at 37 °C for 30 minutes, and just then, the resulting complexes are washed with 0-lx SELEX buffer, distilled water or 0-500 mM EDTA by centrifugation or filtration through a 0.42-mm membrane so as to remove unbound target probes and target probes of low affinity, before the complexes are used in the second reaction. If the specific substances are proteins, the single-stranded nucleic acid-protein complexes and unbounded single-stranded nucleic acids may be separated by filtration through a 0.45-mm nitrocellulose membrane, or adsorption of the protein sample to an ELISA plate.

[56] The step (4) of the present invention is the step of dissociating the probes from the specific substance-probe complexes isolated by washing. The specific substance-probe complexes obtained in the step (3) are treated in a suitable solution at 60-100 °C for 5-10 minutes so as to dissociate the probes from the complexes.

[57] The binding extent between the specific substances and the probes is determined depending on the specificity and binding affinity therebetween, and from this binding extent, the specific substances can be identified and quantified. The specificity and binding affinity between the specific substance recognition sites of the probes and the specific substances determine the binding extent of the specific substance-probe complexes, and the amount of the specific substances determines the amount of the specific substance-probe complexes. The probes dissociated from the complexes are determined by the amount of the specific substance-probe complexes, and the probe analysis is performed by a nucleic acid analysis technique. Thus, the analysis of the nucleic acid analysis results allows the kind and quantitative change of the specific substances to be found.

[58] The step (5) of the present invention is the step of investigating the dissociated probes and can be performed by various methods as shown in FIGS. 11 and 12 depending on the kind of the probes.

[59] FIG. 11 shows a measurement method based on a PCR technique, which comprises reacting E. coli with the probes, isolating the E. coli-probe complexes by filtration, dissociating the probes from the complexes, amplifying the dissociated probes with forward and reverse primers corresponding to the immobilization sites of the probes, and measuring the amplification products by various techniques, including elec- trophoresis.

[60] In the method shown in FIG. 12, E. coli is reacted with labeled probes, and the E. coli-probe complexes are isolated by filtration. The probes are dissociated from the complexes, and the dissociated probes are placed in a fluorescence measurement tube. The tube is mounted on a fluorometer, and light irradiated from a lamp is irradiated to the tube through an excitation filter. Fluorescence emitted from the tube allows analysis of the specific substances by a fluorescence value from an emission filter.

[61] The above method is useful in a system capable of investigating and analyzing biomolecules, including microorganisms, cells and proteins.

[62] In still another aspect, the present invention provides an analysis kit for the identification and quantitative analysis, comprising: single-stranded nucleic acid probes having base sequences specifically binding to E. coli; an analytic reagent containing single-stranded nucleic acid or 0.2% BSA, which is used in the binding reaction between the probes and the E. coli strains; a membrane for the isolation of E. coli- probe complexes; a solution for use in the dissociation of the E. coli-probe complexes; and forward and reverse primers.

[63] Also, the present invention provides a system for investigating and analyzing biomolecules, including microorganisms, cells and proteins, in a sample, using single- stranded nucleic acid probes.

[64] The system for the investigation and analysis of biomolecules comprises the inventive single-stranded nucleic acid probes, an analytical reagent, and a means for investigating the probes.

[65] The biomolecules include microorganisms, such as bacteria and virus, cell lines, proteins or enzymes isolated therefrom.

[66] The single-stranded nucleic acid probe means single-stranded nucleic acid consisting of either immobilization site-specific substance recognition site- immobilization site or fluorescent substance-specific substance recognition site. The analytical reagents include a selection buffer, a selection buffer containing 0.2% bovine serum albumin (BSA) or an analytical reagent containing single-stranded nucleic acid. The investigation of the signaling substances may be performed by a nuc leic analysis technique, and the obtained data can be analyzed by a bioinformational method, and preferably a PCR analysis technique. The identification and analysis system can be used to analyze the biomolecules.

[67]

[68]

[69] Advantageous Effects

[70] As described above, the present invention provides the method for identifying the specific substances and analyzing the quantitative change of the specific substances, using the dual function of the single-stranded nucleic acid aptamers which form complexes with the specific substances in a given environment or form double- stranded nucleic acids with complementary single-stranded nucleic acids. The inventive method can be used in the investigation and analysis of biomolecules, including microorganisms, cells and proteins, and ultimately, it can be widely applied in medicine, veterinary, environmental engineering, food engineering, agricultural industry, and the like.

[71] Best Mode for Carrying Out the Invention

[72] It will be apparent to these skilled in the art that various modification and variation can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention. The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

[73] Mode for the Invention

[74] Hereinafter, the present invention will be described in further detail by examples. It is to be understood, however, that these examples are provided for illustrative purpose only and are not construed to limit the scope of the present invention. [75] Example 1 : Selection of single-stranded nucleic acids

[76] A SELEX library, primers and single-stranded nucleic acids were prepared in the following manner.

[77] As shown in Tables 1 and 2 below, an HPLC-purified SELEX library consisted of base sequences in which 60 nucleotides are randomly arranged between primer binding sequences each consisting of 18 nucleotides (5'-A CCA GCT TAT TCA ATT (N)60 AGA TAG TAA GTG CAA TCT-3' MWG-Biotech AG). A forward primer set forth in SEQ ID NO: 1 (5'-ATA CCA GCT TAT TCA ATT-3') and a reverse primer set forth in SEQ ID NO: 2 (5'-AGA TTG CAC TTA CTA TCT-3') were used as primers in PCR (polymerase chain reaction) for the synthesis of double-stranded or single stranded DNA. Meanwhile, as the immobilization sites (X and Y) of the inventive single- stranded nucleic acid probes, an X sequence set forth in SEQ ID NO: 27 (5'- ATA CCA GCT TAT TCA ATT-3') and an Y sequence set forth in SEQ ID NO: 28 (5'-AGA TAG TAA GTG CAA TCT-3') were used.

[78] The selection of single-stranded nucleic acid aptamers to target substances was performed by the standard SELEX method (Bock L.C. et al., Nature, 355f636θL pp564-6, 1992). The aptamers are oligonucleotides showing specific binding affinity to the specific substances, and highly specific base sequences can be obtained by repeating the selection processes.

[79] The single-stranded nucleic acids synthesized above were heated in a selection buffer (50 mM Tris-HCl (pH 7.4), 5 mM KC1, 100 mM NaCl, 1 mM MgCl , 0.1% NaN ) at a concentration of 1015 bases/ml and 200 pmol/200 ml from the next time, at 80 °C for 10 minutes, followed by standing for 10 minutes. Five-fold (relative to the used single-stranded nucleic acids) yeast tRNA (Life Technologies) and 0.2 % BSA (bovine serum albumin, Merck) were added to prepare a reaction solution. Single- stranded nucleic acids were added to and reacted with a selection buffer containing 106 E. coli at 37 °C for 30 minutes. Single-stranded nucleic acids binding to E. coli were isolated by centrifugation and washed with 1 ml of a selection buffer (containing 0.2% BSA). The reaction product was treated with solution I (selection buffer, lOmM EDTA) to isolate single-stranded nucleic acids, and the single-stranded nucleic acids were completely isolated by phenol extraction and ethanol extraction. In the second process, the single-stranded nucleic acids resulting from the ethanol precipitation were subjected to asymmetric PCR with forward and reverse primers. The single-stranded nucleic acids subjected to the same process as describe above.

[80] The SELEX process was repeated 8 times, and then, the finally isolated single- stranded nucleic acids were subjected to PCR with forward and reverse primers, thus synthesizing double-stranded nucleic acids. The PCR products were cloned into a T- vector and their base sequences were determined. Then, single-stranded nucleic acids El 8 having high binding affinity to E. coli were selected (SEQ ID NO: 20; Korean Patent Application No. 2002-0064027).

[81] [82] Table 1

[83] [84]

[85] [86] Example 2: Fabrication of single-stranded nucleic array [87] Single-stranded nucleic acid capture probes to be immobilized on the surface of a glass slide were prepared by subjecting a plasmid having the selected single-stranded nucleic acids cloned therein to the standard PCR using 5 '-primers and 3 '-primers modified with an amine group at the 5 '-end. A solution comprising 1 pg isolated plasmid, PCR reaction solution, 100 pM 5 '-primer, 100 pM 3 '-primer, and dNTP mixture (5mM dATP, 5mM CTP, 5mM dGTP, 5 mM dTTP) was subjected to the standard PCR, thus synthesizing double-stranded nucleic acids. The PCR reaction was performed 30 cycles each consisting of 30 sec at 94 °C, 30 sec at 52 °C, and 20 sec at 72 °C. Unbound primers were removed according to the protocol of QIAquick PCR Purification Kit (Qiagen) so as to prepare capture probes to be immobilized on a glass slide. As the glass slide, aldehyde-coated CSS (BMS Co.) was used to fabricate an array having the single-stranded nucleic acids arranged thereon, as shown in FIG. 8. The fabrication of the array was performed using a pin-type Microarrayer system (GenPak), and the spot center-to-center spacing was 370 mm. Each of the single- stranded nucleic acids was dissolved in 3x SSC buffer to adjust the concentration. The humidity within the arrayer was maintained at 70% to perform the spotting. The spotted slides were left to stand in a humidified chamber for 15 minutes, and then, baked at 80 °C for 2-4 hours. Immediately after the single-stranded nucleic acids were immobilized on the glass slide by a known method, and the slide was dried by centrifugation and then stored in a light-shielded condition.

[88]

[89] Example 3: Preparation of specific substance-target probe complexes

[90] Single-stranded nucleic acids used as target probes were prepared by performing asymmetric PCR on plasmid templates having the single-stranded nucleic acids E1-E18 and N1-N6 (selected by SELEX) cloned therein using Cy5 (sulfoindocyanine)-labeled 5'-primer (SEQ ID NO: 1), and 3'-primer (SEQ ID NO: 2). A solution comprising 1 pg isolated plasmid and a PCR reaction solution containing 10 pM Cy5-labeled 5'-primer, 1 pM 3'-primer, and dNTP mixture (5mM dATP, 5mM CTP, 5mM dGTP, 5 mM dTTP) was subjected to the standard asymmetric PCR so as to label the single-stranded nucleic acids with the fluorescent substance. The PCR was performed for 30 cycles each consisting of 30 sec at 94 °C, 30 sec at 52 °C, and 20 sec at 72 °C. Unbound primers and bases were removed according to the protocol of QIAquick PCR Purification Kit (Qiagen).

[91] A sample of specific substances was prepared by diluting E. coli, and the colony forming unit (cfu) of E. coli was measured.

[92] Specifically, the purified target probe solution was mixed with 150 ml selection buffer (containing 0.2% BSA) containing the sample, and the mixture was allowed to react at 37 °C for 30 minutes. The E. coW-target probe complexes were washed three times with selection buffer or 50 mM EDTA by centrifugation, and the reaction product was collected by centrifugation at 15,000xg for 5 minutes. The collected product was added to a hybridization solution and heated at 95 °C for 5 minutes, followed by treatment with ice.

[93]

[94] Example 4: Isolation of complexes and reaction between capture probes and target probes

[95] The glass slide prepared in Example 2, which has the single-stranded nucleic acids attached thereto, was treated with a hybridization solution and allowed to react with the solution at 42 °C for 30 minutes. The hybridization solution contained 1 M sodium chloride, 0.3 M sodium citrate, 0.5% SDS or 100 mg/ml salmon sperm DNA, and 0.2% bovine serum albumin or single-stranded nucleic acid. The prehybridized glass slide was treated with the solution prepared in Example 3, and hybridized with the solution at 42 °C for 12 hours, and the array was washed with a washing solution. The washing was performed with IX SSC + 0.2% SDS, IX SSC + 0.2% SDS, 0.5X SSC + 0.2% SDS, and then 0.01X SSC + 0.2% SDS, at 42 °C for 30 minutes for each washing step.

[96]

[97] Example 5: Investigation and analysis of spots on array

[98] After completion of the washing in Example 4, the glass slide was dried by centrifugation and then investigated using a laser scanner (GenePix4000, Axon) having a suitable wavelength (635 nm) to excite the used fluorescent dye (Cy5). The fluorescent images were stored in a multi-image-tagged image file (MIFF) format and then analyzed with suitable image analysis software (GenePix Pro 3.0, Axon) (FIG. 3). The signal intensity (quanta) per spot was used exclusive of a background signal which means a local background consisting of the surrounding signals of four spots. Generally, when at least 90% of pixels in a spot show a higher signal intensity than background signal + 2 standard deviation (S.D.), the spot is included in data analysis, and when a spot does not satisfy the above condition, it is classified as a bad spot and not included in data analysis. Variation with labeling efficiency is normalized using the signal of the internal standard (IS) (e.g., Normalized Intensity = Probe Intensity/IS intensity), and in the case of monolabeling, the signal intensity of the Cy5 channel is recorded as a result, and if the spotting is made two times, the average value is used. With respect to the signal strength (S) of the probes, the signal intensity of each of pixels in a spot is calculated and the median value of pixel-by-pixel is used. The deviation of the signal intensity (S) according to labeling efficiency is normalized using the internal standard.

[99] S' (normalized value) = S (Cy5-reference) x (Cy5-IS)

[100] As described above, by plotting the analysis result for the pixel density and the amount of the sample, the number of E. coli in the sample can be quantified from the array fluorescent data. The pattern of the spots can be analyzed by, for example, clustering.

[101] The fluorescence varies depending on spots by double strands formed by the capture probes and the target probes. The binding extent and amount between the specific substances and the target probes are determined depending on the specificity and binding affinity therebetween. The base sequences of the target probes dissociated from the specific substance-target probe complexes and the capture probes influence the stability of the target probe-capture probe double strands, and the amount of the target probes influence the fluorescence. Thus, by analyzing the fluorescence of the formed spots, the kind and quantitative change of the specific substances can be analyzed.

[102] The results of the tests are shown in FIGS. 9 and 10. As shown, in the glass slide having the single-stranded nucleic acids attached thereto, sites to which aptamers to E. coli (E1-E18, SEQ ID NOS: 3-20) have been attached showed fluorescence, but the negative controls (N1-N6, SEQ ID NOS: 21-26) showed no fluorescence. Also, Listeria monocytogenes (ATCC No. 19118) was used as a sample and tested in the same manner as described above, and as a result, fluorescence could not be detected. From the above results, it could be seen that the base sequences of El -El 8 (SEQ ID NOS: 3-20) selected by SELEX had a specific binding affinity to E. coli.

[103] Also, the case of 100 cfu of E. coli (FIG. 9) was compared with the case of 10,000 cfu of E. coli (FIG. 10) and as a result, it could be seen that the fluorescence was higher in the case of 10,000 cfu of E. coli than the case of 100 cfu of E. coli, indicating that the difference in fluorescence had a correlation with the cfu of E. coli. Namely, the capture probe-target probe double strands were formed, and the fluorescence varied depending on the number of the double strands, had a correlation with the number of microorganisms, and the use of the fluorescence allowed the number of microorganisms to be determined.

[104] As described above, the use of the array to which the single-stranded nucleic acids having recognition sites for various substances have been attached allows the identification of various substances in one sample and the investigation and analysis of the quantative change of the substances.

[105]

[106] Example 6: Construction of probes

[107] Probes were constructed in view of nucleic acid analysis methods. In the case of a PCR technique, the probes were prepared by performing the standard asymmetric PCR on plasmid templates having the selected single-stranded nucleic acids cloned therein using forward and reverse primers. For this purpose, a solution comprising 1 pg isolated plasmid and a PCR reaction solution containing 10 pM forward primer, 1 pM reverse primer and dNTP mixture (5mM dATP, 5mM CTP, 5mM dGTP, 5 mM dTTP) was subjected to the standard asymmetric PCR so as to construct probes. The PCR reaction was performed for 30 cycles each consisting of 30 sec at 94 °C, 30 sec at 52 °C and 20 sec at 72 °C. According to the protocol of QIAquick PCR Purification Kit (Qiagen), unbound primers were removed to finally prepare probes. In the case of fluorometry, probes were prepared by labeling the 5 '-end of the selected aptamers with Cy-3 and purifying the labeled aptamers by HPLC.

[108]

[109] Example 7: Preparation of specific substance-probe complexes [110] Reaction was performed in three steps of the first reaction, the second reaction and the third reaction. A sample was prepared by diluting E. coli (Promega), and the colony forming unit (cfu) of E. coli in the sample was measured according to the standard method (Sambrook J., Fritsh E. F., and Maniatis T. 1989. Molecular cloning (2nd edition). Cold spring Harbor Laboratory Press. 3:A.1-A.6).

[Ill] In the first reaction, the purified probe solution was mixed with 150 ml selection buffer (containing 0.2% BSA) containing the sample, and the mixture was allowed to react at 37 °C for 30 minutes. In the second reaction, the E. coli-probe complexes were washed with three times with selection buffer by centrifugation, and the reaction product was collected by centrifugation at 15,000xg for 5 minutes. Alternatively, the reaction solution was filtered through a 0.45 mm nitrocellulose membrane or a PVDF (polyvinylidiene fluoride) filter and washed three times with selection buffer. In the third reaction, for the centrifugation, 1,000 ml of distilled water was added to the pellets containing the specific substance-probe complexes, and for the filtration, the membrane was placed in 1,000 ml of distilled water and treated at 60-100 °C for 5-10 minutes, and then, the solution was collected.

[112]

[113] Example 8: Investigation and analysis of probes dissociated from specific substance-probe complexes

[114] 8-1: Investigation and analysis by PCR technique

[115] In order to analyze the probes dissociated in Example 7 by a PCR technique, double-stranded nucleic acids were synthesized by performing PCR with forward and reverse primers. For this purpose, a solution comprising the dissociated probes, PCR reaction solution, 100 pM forward primer, 100 pM reverse primer, and dNTP mixture (5mM dATP, 5mM CTP, 5mM dGTP, 5 mM dTTP), was subjected to the standard PCR so as to synthesize double-stranded nucleic acids. The PCR reaction was performed for 8 cycles each consisting of 30 sec at 94 °C, 30 sec at 52 °C and 20 sec at 72 °C. The PCR products were electrophoresed on 2% agarose gel.

[116] As a result of the electrophoresis, band intensity was in proportion to the amount of the E. coli sample. By the specificity and binding affinity between E. coli and the E. coli recognition sites of the probes, the binding extent between E. coli and the probes is determined, and by the amount of E. coli, the binding amount therebetween is determined. The amount of the probes dissociated from the E. coli-probe complexes determines the amount of the PCR products. Thus, the analysis of intensity of the formed bands allowed E. coli and the quantitative change of E. coli to be confirmed.

[117] The test results are shown in FIG. 13. As shown, in the groups of reaction between E. coli and the probes, bands were formed. Also, Listeria monocytogenes (ATCC No. 19118) was used as a sample and tested in the same manner as described above and as a result, bands could not be detected. Moreover, the case of 100 cfu of E. coli was compared with the case of 10,000 cfu of E. coli and as a result, it could be seen that the band intensity was higher in the case of 10000 cfu of E. coli than the case of 100 cfu of E. coli, indicating that a difference in the band intensity has a correlation with the cfu of E. coli. Namely, the E. coli-probe complexes were formed, and thus, the band intensity varied depending on the number of E. coli and had a correlation with the number of microorganisms, and the use of the band intensity allowed the number of microorganisms to be determined. Limits of detection in detecting microorganisms using aptamers and PCR technology can generally reach 1 cfu of microorganisms, but 100 cfu in this Example.

[118]

[119] 8-2: Investigation and analysis by fluorometry

[120] In order to analyze the dissociated probes by a fluorometer, fluorescence was measured with a fluorometer (Turner Biosystem, Picofluor , Handheld Dual Channel Fluorometer). The device was set to 0 for a test group containing only the E. coli sample and set to 999 for a test group containing the sample and the same amount of the probes, and then, a comparative group which is a reaction product of the sample and the probes was measured.

[121] The results of the fluorometric analysis showed that the fluorescence varied in proportion to the amount of the specific substances. Depending on the specificity and binding affinity between E. coli and the probes, the binding extent between E. coli and probes are determined, and depending on the amount of E. coli, the amount of binding therebetween is determined. The amount of the probes dissociated from the E. coli - probe complexes influences fluorescence. Thus, the analysis of fluorescence allows E. coli and the quantitative change of E. coli to be confirmed.

[122] The test results are shown in FIG. 14. As shown, in the groups of reaction between E. coli and the probes, the fluorescence was detected. The case of 100 cfu of E. coli was compared with the case of 10,000 cfu of E. coli and as a result, it could be seen that the fluorescence was higher in the case of 10,000 cfu of E. coli than the case of 100 cfu of E. coli, indicating that a difference in the fluorescence has a correlation with the cfu of E. coli. Namely, the E. coli-probe complexes were formed, and the fluorescence varied depending on the number of E. coli and had a correlation with the number of microorganisms, and the use of fluorescence allowed the number of microorganisms to be determined. Limits of detection in detecting microorganisms using aptamers and PCR technology can generally reach 1 cfu of microorganisms, but 100 cfu in this Example.

[123] As described above, the use of the probes having recognition sites for various substances allows the identification of various substances in one sample and the in- vestigation and analysis of the quantitative change of the sample.

[124]

[125] Example 9: Selection of aptamers using mixed sample

[126] 9-1: SELEX of mixed sample

[127] Serum proteins were labeled with biotin and captured with streptoavidin-magnetic beads. A RNA library for SELEX was reacted with the modified serum proteins, and then, the serum protein-RNA complexes were magnetically isolated so as to select RNAs binding to the serum proteins. The isolated complexes were subjected to RT- PCR so as to amply and construct a DNA pool indicating RNAs binding to the serum proteins. The DNA pool was transcribed in vitro to synthesize RNAs, and the selection and amplification processes were performed about 10 times. Then, the obtained RNA pool was subjected to RT-PCR, and the obtained PCR product products (RNAs) were cloned into plasmids, thus constructing an aptamer library for the serum proteins.

[128]

[129] 9-2: Analysis of specific substances in mixed sample

[130] From the aptamer library for the serum proteins, 1,000 clones were selected and subjected to PCR using a forward primer labeled with an amine group at one end and a reverse primer. The PCR products were spotted on a substrate by a microarrayer so as to fabricate a DNA array having 1,000 spots. The DNA array was named an "aptamer- based biochip" 10 ml of a serum sample was added to 90 ml of PBS solution, and a nitrocellulose membrane disc was placed in the serum sample solution and allowed to react for 30 minutes with shaking. The disc having the serum sample attached thereto was treated and reacted with 1,000 RNA aptamers fabricated from the selected clones, for 30 minutes. The disc having the aptamers and serum sample attached thereto was treated with RT-PCR solution and subjected to RT-PCR while labeling with Cy-5. According to the same manner as described above, a Cy-3-labeled standard solution was prepared. The two solutions were mixed with each other at the same ratio, and the fabricated aptamer-based biochip was hybridized with the mixed solution at 60 °C for 4-12 and washed with O.lx SSC solution at 42 °C. After completion of the reaction, images from the aptamer-based biochip were produced by a laser scanner. Images obtained by the analysis of the serum sample of a cardiovascular disease patient and the serum sample of a healthy person are shown in FIG. 15.

[131] As can be seen in FIG. 15, proteins corresponding to reddish spots are much more present in the serum of the cardiovascular disease patient than the healthy person, bluish spots are opposed to that, and yellowish spots are present in both the cases at a similar level.

[132]

Claims

[1] A method for identifying and quantifying specific substances a single-stranded nucleic acid array, the method comprising the steps: (1) immobilizing single-stranded nucleic acid capture probes each consisting of reactive group (X)-spacer site (R)-target probe recognition site (V) on a solid substrate so as to fabricate an array; (2) reacting specific substances with target probes by treatment with an analytical reagent so as to prepare specific substance-target probe complexes; (3) washing and isolating the specific substance-target probe complexes prepared in the step (2); (4) dissociating the target probes from the isolated complexes or amplifying and labeling the target probes; (5) reacting the capture probes with labeled target probes; and (6) investigating substances labeled to the target probes.

[2] The method of Claim 1, wherein the analytical reagent in the step (2) is a SELEX selection buffer containing: (1) 20-100 mM Tris-HCl (pH 7.4), (2) 0-200 mM of each of potassium chloride, sodium chloride or magnesium chloride, and a mixture thereof, (3) 0.05-0.2% sodium azide, and (4) 0.1-0.3% bovine serum albumin or nucleic acid for inhibiting non-specific binding.

[3] The method of Claim 2, wherein the analytical reagent contains 50 mM Tris-HCl (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride or lmM magnesium chloride, 0.1% sodium azide, 0.2% bovine serum albumin or single- stranded nucleic acid.

[4] The method of Claim 1, wherein the specific substance in the step (2) is at least one selected from the group consisting of bacteria, fungi, virus, cell lines, tissue, proteins isolated therefrom, carbohydrates, lipids, polysaccharides, gly- coproteins, hormones, receptors, antigens, antibodies, and enzymes.

[5] The method of Claim 1, wherein the washing buffer in the step (3) is 0-lx SELEX buffer or 0-500 mM EDTA solution.

[6] The method of Claim 1, wherein a nucleic acid hybridization solution in the step (4) comprises: (1) 0.5-1.5 M sodium chloride, (2) 0 -1.0 M sodium citrate, (3) 0.1-0.3% bovine serum albumin, 0-1.0% SDS, 0-10-fold Denhart solution or 0-1% nonfat dried milk for inhibiting non-specific binding, (4) nucleic acid for inhibiting non-specific binding, (5) 30-60 % (v/v) formamide.

[7] The method of Claim 6, wherein the nucleic acid hybridization solution contains 1 M sodium chloride, 0.3 M sodium citrate, 0.5% SDS or 100 mg/ml salmon sperm DNA, and 0.2% bovine serum albumin or single-stranded nucleic acid.

[8] An analysis kit for the identification and quantitative analysis of E. coli, comprising: a single-stranded nucleic acid array fabricated with capture probes having base sequences specifically binding to E. coli; an analytical reagent containing single-stranded nucleic acid or 0.2% BSA, which is used in the binding reaction between target probes and the capture probes; a hybridization solution which is used in the binding reaction between the target probes and the capture probes; and a 5 '-primer and 3 '-primer labeled with Cy-5.

[9] A method for identifying and quantifying specific substances using single- stranded nucleic acid probes, the method comprising the steps of: (1) constructing single-stranded nucleic acid probes each consisting of either immobilization site (X)-specific substance recognition site (V)-immobilization site (Y) or fluorescent substance (F)-specific substance recognition site (V); (2) reacting the probes constructed in the step (1) with specific substances by treatment with an analytical reagent so as to prepare specific substance-probe complexes; (3) washing and isolating the specific substance-probe complexes prepared in the step (2); (4) dissociating the probes from the isolated complexes; and (5) investigating the dissociated probes.

[10] The method of Claim 9, wherein the analytical reagent in the step (2) is a SELEX selection buffer containing: (1) 20-100 mM Tris-HCl (pH 7.4), (2) 0-200 mM of each of potassium chloride, sodium chloride or magnesium chloride, and a mixture thereof, (3) 0.05-0.2% sodium azide, and (4) 0.1-0.3% bovine serum albumin or nucleic acid for inhibiting non-specific binding.

[11] The method of Claim 10, wherein the analytical reagent contains 50 mM Tris- HCl (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride or 1 mM magnesium chloride, 0.1% sodium azide, and 0.2% bovine serum albumin or single-stranded nucleic acid.

[12] The method of Claim 9, wherein the specific substance in the step (2) is at least one selected from the group consisting of bacteria, fungi, virus, cell lines, tissue, proteins isolated therefrom, carbohydrates, lipids, polysaccharides, gly- coproteins, hormones, receptors, antigens, antibodies, and enzymes.

[13] The method of Claim 12, wherein the specific substance is E. coli.

[14] The method of Claim 9, wherein the washing buffer in the step (3) is 0-lx SELEX buffer or 0-500 mM EDTA solution. [15] The method of Claim 9, wherein the investigation in the step (5) is performed by a polymerase chain reaction (PCR) or fluorometric technique. [16] An analysis kit for the identification and quantitative analysis of E. coli, comprising: single-stranded nucleic acid probes having base sequences specifically binding to E. coli; an analytical reagent containing single-stranded nucleic acid or 0.2% BSA, which is used in the binding reaction between the probes and E. coli; a membrane for isolating E. co -probe complexes; a solution which is used in the dissociation of the E. coli-probe complexes; and forward and reverse primers. [17] A method for selecting aptamers from a mixed sample, comprising the steps of: (1) performing SELEX using a mixed sample; (2) constructing an aptamer library using the aptamer pool obtained by SELEX; (3) constructing a DNA array using single-stranded nucleic acids complementary to the base sequences of aptamers selected from the aptamer library; (4) reacting a mixed sample with the selected aptamer pool; (5) isolating the complexes and amplifying the aptamers of the complexes; (6) hybridizing the amplified nucleic acids with the DNA array; and (7) imaging and analyzing the hybridization results.