METHOD FOR OBTAINING PROFILE OF TARGET MOLECULE POPULATION OF SAMPLE

Abstract

The present invention discloses a profiling technique for a target molecule population in a sample including an unknown target molecule, using an aptamer. In the method of the present invention, the target molecule population in the sample may be provided as an aptamer profile including an unknown target molecule, and this aptamer profile can be used to determine whether drug prescription is appropriate (i.e., anticancer drug companion diagnosis, etc.), to provide disease diagnosis information, to monitor drug treatment, to determine drug compliance, to determine the degree or absence/presence of in vitro cellular response to drug treatment, and to obtain useful information to humans for classification or identification of species, etc.

Claims

1. A method of generating a profile for a target molecule population in a sample, the method comprising: (a) treating an aptamer library tagged with a first tag that is specific to a target molecule population in a sample and is capable of binding to a first capture component, with a solid support to which the first capture component is coupled so that the first capture component and the first tag are bound to each other, thereby immobilizing the aptamer library on the first solid support; (b) treating an analysis target sample that is the same kind as the sample, with the first solid support on which the aptamer library is immobilized so that each target molecule of the target molecule population in the analysis target sample and each aptamer of the aptamer library form a target-aptamer complex, thereby obtaining a complex population; (c) isolating the complex population in a state in which the complex population is immobilized on the first solid support by excluding unbound target molecules; (d) attaching a second tag capable of binding to a second capture component to the target molecule of each complex of the isolated complex population; (e) treating the complex population tagged with the second tag, with a second solid support to which the second capture component is coupled so that the second capture component and the second tag are bound and thus the complex population is immobilized on the second solid support; (f) isolating an aptamer population from the complex population immobilized on the second solid support in a form in which the aptamer population remains immobilized on the first solid support; and (g) generating an aptamer profile by quantifying each aptamer of the aptamer population that remains immobilized on the first solid support.

2. The method of claim 1, wherein the profile for the target molecule population in the sample is an aptamer profile.

3. The method of claim 1, wherein the aptamer profile is used to determine whether drug prescription is appropriate, provide disease diagnosis information, monitor drug treatment, determine drug adherence, determine whether there is a cellular response to drug treatment in vitro, determine a degree of a cellular response to drug treatment in vitro, to perform species classification, or identify a species.

4. The method of claim 1, wherein the target molecule is a protein, a glycoprotein, a lipoprotein, or a peptide.

5. The method of claim 1, wherein the target molecule population includes a target molecule that is unidentified.

6. The method of claim 1, wherein the aptamer library specific to the target molecule population in the sample is obtained by: (i) preparing a single-stranded nucleic acid library having different random sequences, thereby having potential binding ability to various target molecules; (ii) reacting the single-stranded nucleic acid library with the target molecule population in the sample to induce specific binding between each single-stranded nucleic acid and each target molecule to form a complex population; (iii) isolating the complex population by excluding unbound single-stranded nucleic acids, and (iv) amplifying the single-stranded nucleic acids of the complex population.

7. The method of claim 1, wherein steps (i) to (iv) are performed once, the excluding of the unbound single-stranded nucleic acids is performed through a washing step, and the washing step is repeated two or more times, and the washing step is performed using a washing buffer containing a surfactant, a salt, a competitor molecule, or a mixture thereof.

8. The method of claim 1, wherein the aptamer is single-stranded RNA or single-stranded DNA.

9. The method of claim 8, wherein the single-stranded RNA or single-stranded DNA comprises nucleotides modified from sugar, phosphate, or base.

10. The method according to claim 1, wherein the sample is a biological sample or a processed sample thereof.

11. The method of claim 1, wherein the first tag and the second tag are biotin or an analog thereof, and the first capture component and the second capture component are streptavidin or an analog thereof.

12. The method of claim 1, wherein the first solid support is a magnetic bead and the second solid support is a non-magnetic support.

13. The method of claim 1, wherein when the target molecule and the aptamer form the complex in step (b), the competitor molecule and the sample are treated together on the first solid support to improve the specific binding between the target molecule and the aptamer.

14. The method of claim 1, wherein the isolating of the aptamer population from the complex population immobilized on the second solid support in step (f) in a form in which the aptamer population is immobilized on the first solid support comprises: heating, treatment with a chaotropic salt, inducing pH change to strong acidity or strong basicity, treatment with a surfactant, or a combination thereof.

15. The method of claim 1, wherein the quantification of the aptamer in step (g) is performed by next-generation sequencing technology, microarray method, or multiple real-time PCR method.

16.-46. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0094] FIGS. 1, 2, 3, 4, and 5 are results showing that when using an RNA aptamer specific to a protein molecule population of the serum of a normal person, and using an aptamer library obtained for a protein molecule population in a serum sample of a lung cancer patient and an aptamer library obtained fora protein molecule population in a serum sample of a normal person, the two samples are distinguished.

[0095] FIGS. 6, 7, 8, 9, and 10 are results showing that when using an DNA aptamer specific to a protein molecule population of a serum, and using an aptamer library obtained for a protein molecule population in a serum sample of a lung cancer patient and an aptamer library obtained for a protein molecule population in a serum sample of a normal person, the two samples are distinguished.

DETAILED DESCRIPTION

[0096] Hereinafter, the present invention will be described in detail with reference to some examples. However, the scope of the present invention is not limited to the examples

<Example 1> Preparation of Reagent and Random Double-Stranded Nucleic Acid Library

<Example 1-1> Reagent

[0097] HEPES, NaCl, KCl, EDTA, EGTA, MgCl.sub.2, and Tween-20 were purchased from Fisher Scientific. Dextran sulfate sodium salt (DxSO4) was purchased from American International Chemical. KOD polymerase (KOD exo(-) polymerase) was purchased fromAvantor's VWR.

[0098] Tetramethylammonium chloride and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) were purchased from Sigma-Aldrich, and streptavidin phycoerythrin (SAP E) was purchased from Moss Inc. 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride and 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF) were purchased from Gold Biotechnology. Streptavidin coated 96-well plates (Pierce streptavidin coated plates HBC, clear 96 wells, product number 15500 or 15501) were purchased from Thermo Scientific. NHS-PEG4-Biotin (EZ-Link NHS-PEG4-Biotin, product number 21329) was purchased from Thermo Scientific, dissolved in anhydrous DMSO, and stored frozen in single-use aliquots. Yeast tRNA was purchased from Life Technologies.

[0099] PCR-grade natural and unnatural nucleotides were purchased from Thermo Fisher Scientific.

[0100] Random oligonucleotides, forward primers, reverse primers, and biotin-coupled forward primers were synthesized through order production by Baonia (in Korea).

[0101] SB18, which is a buffer solution, consists of 40 mM HEPES adjusted with NaOH to pH 7.5, 101 mM NaCl, 5 mM KCl, 5 mM MgCl2, and 0.05% by volume (vN) Tween 20. SB17, which is also a buffer solution, is obtained by adding 1 mM trisodium EDTA to SB18. SB17T, which is a buffer solution, consists of 40 mM HE PE S with pH 7.5, 102 mM NaCl, 5 mM KCl, 5 mM MgCl.sub.2, 1 mM EDTA, and 0.05% Tween 20.

[0102] PB1, which is a buffer solution, consists of 10 mM HE PES adjusted to pH 7.5 with NaOH, 101 mM NaCl, 5 mM KCl, 5 mM MgCl.sub.2, 1 mM trisodium EDTA, and 0.05% by volume (v/v) of Tween-20.

[0103] A CAPSO elution buffer solution consists of 100 mM CAPSO with pH 7.5 and 1 M NaCl. A neutralization buffer solution consists of 500 mM HEPES, 500 mM HCl and 0.05% by volume (v/v) of Tween-20.

[0104] As samples, serum collected at Asan Medical Center (IRB approval number 2015-0607) was used.

<Example 1 2> Preparation of Random Double-Stranded Nucleic Acid Library

[0105] To prepare an RNA aptamer library that specifically binds to a target protein molecule population in the sample, first, single-stranded DNA oligonucleotides (random single-stranded nucleic acids) having the structure represented by General Formula I were prepared by Bioneer (Korea) by order.

TABLE-US-00001 <General Formula 1> 5′-CCACGCTGGGTGGGTCN.sub.40GGACAAAGAGAGAAGAGAAAGAG-3′

[0106] The oligonucleotide having the structure represented by General Formula I consists of a 5 ̆conserved region, a variable region, and a 3 ̆conserved region arranged in this order.

[0107] In the above, the underlined nucleotide sequence is a conserved region that is a fixed region consisting of a known sequence, and N.sub.40, which is a variable region, consists of 40 nucleotides, including bases such as A, G, T, C that occur with the same frequency.

[0108] A double-stranded DNA library was prepared by performing a PCR method using the oligonucleotide of General Formula I as a template. The primers used at this time were the following DS forward primer (SEQ ID NO: 2) containing the T7 promoter sequence and the following DS reverse primer (SEQ ID NO: 3):

TABLE-US-00002 <DS forward primer (SEQ ID NO: 1) 5′-CCACGCTGGGTGGGTC-3′ <DS forward primer with T7 promoter sequence> (SEQ ID NO: 2) 5′-TAATACGACTCACTATAGGGCCACGCTGGGTGGGTC-3′ <DS reverse primer (SEQ ID NO: 3) 5′-CTCTTTCTCTTCTCTCTTTCTCC-3′

[0109] The DS forward primer containing the T7 promoter sequence (SEQ ID NO: 2) includes the T7 promoter sequence (the underlined portion in SEQ ID NO: 2) for RNA polymerase of bacteriophage T7 as well as the primer sequence for the 5′ conserved region (sequence of the underlined portion) of the single-stranded DNA oligonucleotide having the structure represented by General Formula I. The DS reverse primer (SEQ ID NO: 3) is a primer sequence for the 3′ conserved region (sequence of the underlined portion) of the single-stranded DNA oligonucleotide having the structure represented by General Formula I.

[0110] Polymerase chain reaction (PC R) was performed using the forward primer (SEQ ID NO: 2) and the reverse primer (SEQ ID NO: 3), with the single-stranded nucleic acid oligonucleotide of the structure of General Formula I used as a template.

[0111] Specifically, 1,000 pmoles of single-stranded nucleic acid oligonucleotides and 2,500 pmoles of primer pairs (DS forward primer containing a T7 promoter sequence and DS reverse primer) were mixed with 50 mM KCl, 10 mM Tris-Cl (pH 8.3), 3 mM MgCl2, 0.5 mM dNTP (dATP, dCTP, dGTP, and dTTP), and 0.1 U Taq DNA Polymerase (Perkin-Elmer, Foster City Calif.), for the PCR, and then purified with a QIAquick-spin PCR purification column (QIAGEN Inc., Chatsworth Calif.). In this way, double-stranded nucleic acid DNA having a T7 promoter was prepared. This PCR product was a double-stranded DNA containing a T7 promoter (sequence of the underlined portion), and the general structure thereof can be represented by General Formula II below.

TABLE-US-00003 <General Formula II> 5 TAATACGACTCACTATAGGGCCACGCTGGGTGGGTCN.sub.40GGACAAAGAGA GAAGAGAAAGAG-3′

<Example 2> Preparation of Single-Stranded Nucleic Acid RNA Library

[0112] In this example, an RNA single-stranded nucleic acid library to be reacted with a protein molecule population in the sample was prepared. This RNA single-stranded nucleic acid library is a single-stranded nucleic acid library (SSN library) containing 2′-F-substituted pyrimidines which are modified nucleotides. It was synthesized by performing in vitro transcription of RNA single-stranded nucleic acids containing 2′-F-substituted pyrimidines, using the PCR product having the structure of general formula II, with the DuraScribe T7 Transcription Kit (EPICENTER, USA). After the synthesis, purification was performed.

[0113] Specifically, 200 pmoles of the prepared double-stranded DNA having the structure of General Formula II, 40 mM Tris-Cl (pH 8.0), 12 mM MgCl.sub.2, 5 mM DTT, 1 mM spermidine, 0.002% Triton X-100, 4% PEG 8000, 5 U T7 RNA polymerase, and 1 mM ATP and GTP, and 3 mM 2′F-CTP and 2′F-UTP were mixed and reacted at 37 éC for 6 to 12 hours, followed by purification using a Bio-S pin 6 chromatography column (Bio-Rad Laboratories, Hercules Calif.). The amount and purity of the purified nucleic acid were determined with a UV spectrometer.

<Example 3> Preparation of RNA Aptamer Library Specifically Binding to Protein Molecule Population of Sample, and Application Thereof to Sample Classification

<Example 3-1> Preparation of SSN-FSS Library

[0114] (1) Preparation of SSN Library Tagged with First Tag

[0115] First all reagents and enzymes in Table 1 except for 30% PEG and DMSO were thawed on ice. The DMSO was thawed at room temperature, 30% PEG was warmed at 37 éC for 5 to 10 minutes until the volume becomes a liquid.

[0116] 5 ≈ of the SSN library was transferred to a micro-centrifuge tube. After heating the SSN library at 85 éC for 3 to 5 minutes so that the SSN has a linear primary structure, the SSN library was immediately placed on ice.

[0117] Next, a biotin labeling reaction solution for the SSN library was prepared by adding reagents and enzymes in the order listed in Table 1 below.

TABLE-US-00004 TABLE 1 Reaction to ligate first tag to SSN library Volume Final Concentration Component (≈L) (Amount) Nuclease-free Water 3 — 10X RNA Ligase Reaction Buffer 3 1X RNase Inhibitor 1 1.33 U/≈L (40 U) SSN Library 5 1.67 ≈M (50 pmol) Biotinylated Cytidine (Bis)phosphate 1 33.3 ≈M (1 nmol) T4 RNA Ligase 2 1.33 U/≈L (40 U) 30% PEG 15 15% Total 30 —

[0118] For the reaction solution, the last added reagent was 30% PEG. After carefully adding the 30% PEG to the reaction mixture with a pipette, the mixture was blended using a new pipette tip and reacted at 16 éC for 2 hours so that a reaction for ligating the biotin to the SSN was performed.

[0119] Next, 70 ≈ of nuclease-free water was added to the reaction solution, and 100 ≈ of chloroform:isoamyl alcohol was added thereto to extract RNA ligase. The reaction mixture was gently stirred and then centrifugated in a microcentrifuge at high speed for 2 to 3 minutes so that the phases were separated. Carefully, the upper aqueous layer containing biotinylated SSN was taken and transferred to a new tube.

[0120] In this way, a Bio-SSN library in which biotin as a first tag was added to the SSN 3 end of the SSN library was prepared.

[0121] The stock concentration of the prepared Bio-SSN library was 4 nM. The Bio-SSN library stock mix was diluted 4-fold with an SB17 buffer, heated at 95 éC for 5 minutes, and then cooled to 37 éC for 15 minutes before use. Streptavidin-coupled magnetic beads serving as a first support were washed twice with 150 mL of a P B1 buffer before use.

[0122] 2) Preparation of SSN-FSS Library

[0123] 133 ≈ of a suspension of streptavidin-coupled magnetic beads (Streptavidin-Coupled Dynabeads, Thermo Fisher Scientific, USA) serving as a first solid support (FSS) dissolved in 1×SB17, Tw (40 mM HEPES, 102 mM NaCl, 1 mM EDTA, 5 mM MgCl.sub.2, 5 mM KCl, 0.05% Tween-20) was placed in a tube. An about 1.1× Bio-SS N library (with biotin added to the 3 end of the SSN) was thawed by vortexing. The 1.1× Biotin-SSN library was then boiled for 10 minutes, vortexed for 30 seconds, and cooled to 20 éC in a water bath for 20 minutes. Next, 100 ≈ of the cooled 1.1× Bio-SSN library was added to the tube in which the FSS (Streptavidin-Coupled Dynabeads) was contained. The mixture was protected from light and incubated at 25 éC for 20 minutes on a shaker set at 850 rpm so that the biotin portion of the Bio-SSN library and the avidin portion of the capture component of the FSS (Streptavidin-Coupled Dynabeads) were reacted to prepare a suspended SSN-FSS library.

[0124] The tube with the prepared suspended SSN-FSS library was centrifuged to remove the solvent 190 ≈ of a 1× CAPS prewash buffer (50 mM CAPS, 1 mM EDTA, 0.05% Tw-20, pH 11.0) was added, and the suspended SSN-FSS library was shaken for 1 minute. The CAPS prewash buffer was then removed by centrifugation. The CAPS washing and the centrifugation for removal of the CAPS buffer were then repeated once more.

[0125] For purification of the suspended SSN-FSS library, 190 ≈ of 1×SX17, Tw was added to the tube containing the suspended SSN-FSS library, and the suspended SSN-FSS library was then shaken for 1 minute. The 1×SB17, Tw was then removed by centrifugation. In addition, 190 ≈ of the 1×SX17, Tw was added, and the suspended SSN-FSS library was shaken for 1 minute. The 1×SB17, Tw was then removed by centrifugation (for 1 minute at 1000×g).

[0126] 150 ≈ of a storage buffer (150 mM NaCl, 40 mM HEPES, 1 mM EDTA, 0.02% sodium azide, 0.05% Tween-20) was added to the tube containing the suspended SSN-FSS library. Thus, the SSN-FSS bead library in a suspension state was purified. The tube was carefully sealed and stored in darkness at 4 éC until use.

[0127] The solution to be used later was stored for a long time at −20 éC, and on the day of analysis, each suspended SSN-FSS library was thawed at 37 éC for 10 minutes, placed in a boiling water bath for 10 minutes, and cooled to 25 éC for 20 minutes before use.

[0128] After such heating and cooling, in actual use, 55 ul of each 2× suspended SSN-FSS library was manually pipetted into a 96-well PCR plate, and the tube was resealed with foil until use.

<Example 3-2> Preparation of Analysis Sample

[0129] As samples, serums provided by Asan Medical Center (IRB approval number 2015-0607) were used.

[0130] To prevent protein degradation, each of the samples was diluted in 0.94×SB17 containing 0.6 mM MgCl.sub.2, 1 mM Trisodium EDTA, 0.8 mM AEBSF, and yeast tRNA that is 5 times the SSN concentration. Thus, a 10% serum sample solution was prepared and used.

<Example 3-3> Reaction Between SSN-FSS Library and Sample

[0131] 55 .Math.L of the SSN-FSS library solution in a suspended state was added to a tube containing 55 .Math.L of the diluted sample solution. The sample composed of molecule populations and the suspended SSN-FSS library were mixed by pipetting, and the mixture was sealed with a foil cover. Thereafter, the tube was subjected to a reaction to form a complex (M-SSN-FSS) between the protein molecule (M) population of the sample and the SSN-FSS in a processing device at 37 éC for 3 hours and 30 minutes.

[0132] After the completion of the complex formation reaction, the tube containing 100 .Math.L of the reaction mixture was placed on a stand with a magnet, and the liquid layer was carefully removed with a pipette. The tube was washed 4 times with 300 .Math.L of a PB1 buffer containing 1 mM dextran sulfate and 50 .Math.M biotin (to block a first capture component). Then, the tube was washed 3 more times with 300 .Math.L of a PB1 buffer to prepare the M-SSN-FSS complex population.

<Example 3-4> Second Tag Addition, Immobilization on Second Solid Support, and Elution of SS N-FSS Population

[0133] To the tube containing the M-SS N-FSS complex population, 150 .Math.L of a freshly prepared 1 mM NHS-PEG4-biotin solution in a PB1 buffer was added and treated with shaking for 5 minutes, and biotin serving as a second tag was added the molecules of the suspended M-SSN-FSS complexes.

[0134] The tube was centrifuged, the liquid was removed by suctioning, and the tube was washed 8 times with 300 .Math.l of a PB1 buffer supplemented with 10 mM glycerin. 100 .Math.l of a PB1 buffer supplemented with 1 mM dextran sulfate was added.

[0135] The buffer solution containing the second tag-added bio-M-SS N-FSS complex population in suspension in the tube was transferred to the wells of a plate coated with streptavidin serving as a second solid support (SSS). After allowing reaction at 800 rpm at room temperature for 10 minutes, the bio-M-SSN-FSS complex was induced to be immobilized on the second solid support through biotin serving as the second tag, so that an immobilized SSS-M-SSN-FSS complex was prepared.

[0136] Next, the liquid was carefully removed from the wells of the plate with a pipette, and the wells were washed 8 times with 300 .Math.L of a PB1 buffer supplemented with 25% propylene glycol (or 30% glycerol). The wells were washed 5 times with 300 .Math.L of a SB17T buffer, and finally the washing liquid was suctioned.

[0137] 100 .Math.L of a CAPSO elution buffer was added, and the immobilized SSS-M-SSN-FSS complex was shaken for 5 minutes, so that the SS N-FSS complex was eluted. The SSN-FSS complex population was obtained by manually transferring 90 .Math.L of the solution from the wells of the plate to the wells of a PC R plate containing 10 .Math.L of a neutralization buffer.

<Example 3-3> Preparation of RNA Aptamer Library Specifically Binding to Protein Molecule Population of Sample

[0138] F or the SSN-FSS complex population obtained above, RT-PCR and in vitro transcription were performed in the same manner as in Examples 1 and 2 above. Thus, finally, an RNA aptamer library that specifically binds to the protein molecule population of the sample was prepared.

[0139] An RNA aptamer library having higher specificity and affinity may be prepared by additionally performing the procedures of Examples 3-1 to 3-5 on the prepared RNA library at least once. However, since it is ideal that the RNA library specific to the protein molecule population in the sample should reflect the diversity of protein molecules included in the sample as it is, the steps of Examples 3-1 to 3-5 were performed only once, but the washing process was varied and repeated as described above to obtain an RNA aptamer library that well reflects the diversity of protein molecules included in the sample.

<Example 3-6> Application to Differentiation Between Analysis Samples and Comparative Samples

[0140] The RNA aptamer library for the protein molecule population of each of the serum samples of normal persons obtained in Examples 3-5 was applied to classification of six serum samples (analysis samples L5, L7, L8, L10, L13, and L14) derived from lung cancer patients and six serum samples (comparative samples N1 to N6) derived from normal persons.

[0141] As test samples, lung cancer patient serum samples and normal person serum samples provided by Seoul Asan Hospital (IRB approval number 2015-0607) were used. In the analysis, the same procedures as in Examples 3-1 and 3-4 were performed on each sample to isolate an RNA aptamer population specifically binding to a protein molecule population in a test sample and to a protein molecule population in a control sample.

[0142] Specifically, after attaching the first tag to the RNA aptamer library, the first solid support (streptavidin) was reacted with magnetic beads to prepare an RNA aptamer library (SS N-FSS library) bound to the first solid support; and was then reacted with the sample to prepare a complex population (M-SSN-FSS complex population) bound to the protein population in the sample. Next the second tag was attached to the protein molecule of the complex, then reacted in the streptavidin (i.e., second solid support)-coated wells of a plate, and washed. Next; the aptamer population bound to the protein molecule population was eluted in a form (SSN-FSS complex form) bound to the first solid support.

[0143] Using the eluted complex population as a template and using the primers shown in Example 1 above, cDNA was prepared. Next, one-way PCR was performed on the cDNA once to obtain a collective double-stranded DNA fragment and an NGS library was prepared using Ion AmpliSeq Library Kit 20 (Ion Torrent, Thermo Fisher Scientific) according to the manufacturer's protocol. Next, with Ion Torrent (104, Thermo Fisher), the nucleotide sequence of each DNA fragment was analyzed. In addition, the occurrence frequency of each DNA fragment (i.e., the number or amount of the DNA fragments) and an aptamer profile, which is the overall information of the occurrence frequency of the DNA fragments, were obtained.

[0144] The results are shown in FIGS. 1 to 5. FIGS. 1 and 2 show the number of types of binding aptamers, the total occurrence frequency (i.e., total number of binding aptamers), the average frequency, and the maximum frequency in each analysis sample (lung cancer sample) and in each comparative sample (non-lung cancer sample), respectively. FIGS. 3 and 4 show aptamer profiles for some aptamers (BioSign_serial number) in the six lung cancer samples and aptamer profiles for some aptamers in the normal samples, respectively. FIG. 5 shows comparison between aptamer profiles for some aptamers that have a difference in the occurrence frequency in the lung cancer samples and the normal samples.

[0145] Referring to FIGS. 1 to 5, it is seen that the profile (the overall occurrence frequency of each RNA aptamer) of the RNA aptamer library reacted with the analysis target sample and the profile (the overall occurrence frequency of each RNA aptamer) of the DNA aptamer library reacted with the comparative sample are clearly distinguished.

<Example 4> Preparation of DNA Aptamer Library Specifically Binding to Protein Molecule Population of Sample, and Application Thereof to Sample Classification

<Example 4-1> Preparation of DNA Aptamer Library Specifically Binding to Protein Molecule Population of Sample

[0146] A forward primer (Bioneer, Korea) in which the oligonucleotide of <General Formula I> of Example 1 is used as a template and custom-made biotin is conjugated to the 5-end thereof and a reverse primer (Bioneer, Korea) in which biotin is not conjugated were used fora PC R reaction, so that biotin-attached double-stranded DNA was prepared. Here, it is necessary to check whether the amplified product is used or the oligonucleotide is used, as the template.

[0147] First, 10 .Math.M of an oligonucleotide template, 12 .Math.M of 5 ̆a biotin-conjugated primer, 0.5 mM of dNTP, and 0.25 U/mL of KOD polymerase (KOD exo(-) polymerase) were added to in 1×SQ20 buffer to perform a PCR reaction to prepare double-stranded DNA. Next the double-stranded DNA was captured on high-capacity MyOne-streptavidin paramagnetic beads (Invitrogen, catalog number 65001, Invitrogen, USA) serving as the first solid support (FSS) for 2 hours at room temperature, so that a double-stranded DNA-FSS complex library having a random sequence was prepared. It was progressed.

[0148] The prepared double-stranded DNA-FSS complex library was washed several times with a selected buffer (SBT: 40 mM HE PE S, pH 7.4; 102 mM NaCl; 5 mM KCl, 5 mM MgCl.sub.2 0.05% Tween 20). The complex library was suspended on 20 mM NaOH for 5 minutes to elute a modified sense strand. The eluent was neutralized to pH 7.4 with the required volume of 700 mM HCl, 180 mM HE PE S, pH 7.4, 0.45% Tween 20, and then concentrated with an Amicon Ultra-15 (Millipore) centrifugal filter to a residual volume of approximately 250 ≈.

[0149] In this way, a single-stranded DNA-FSS (‘SSN-FSS) complex library having a random sequence was prepared.

[0150] Next, the normal human serum sample prepared in Example 3-2 and 1000 or more (ℏ1000) pmol (˜10.sup.15) of the prepared SSN-FSS complex were mixed with SB17T buffer (40 mM HEPES, pH 7.5, 102 mM NaCl, 5 mM KCl, 5 mM MgCl.sub.2, 1 mM EDTA, 0.05% Tween 20) to form an M-SSN-FSS complex, and were washed with SB17T buffer to remove unbound serum proteins, single-stranded DNA (SS N), and biotin complexes.

[0151] To the tube containing the M-SSN-FSS complex population, 150 .Math.L of a 1 mM NHS-PEG4-biotin solution freshly prepared in a PB1 buffer was added, and the tube was shaken for 5 minutes, so that biotin serving as a second tag was added to the molecules of the suspended M-SSN-FSS complexes. The addition of the biotin to the protein molecule can be achieved by covalent bonding between NHS-PEG4-biotin (Thermo Scientific, Pittsburgh, Pa., catalog no. 21329) and a lysine residue according to the manufacturer's protocol. Specifically, the M-SSN-The FSS complex population (300 pmol in 50 ≈) was exchanged with a Sephadex G-25 MicroSpin column provided with a SB17T buffer (40 mM HEPES, pH 7.5, 102 mM NaCl, 5 mM KCl, 5 mM MgCl.sub.2, 1 mM EDTA, 0.05% Tween 20). The NHS-PE G4-biotin was added so as to become 30 WI, and the reactant was reacted at 4 éC for 16 hours to prepare a biotin-added complex, i.e., bio-M-SSN-FSS. Unreacted NHS-PEG4-biotin was removed with a Sephadex G-25 MicroSpin column.

[0152] The buffer solution containing the second tag-added bio-M-SS N-FSS complex population that was in a suspension state in the tube was transferred to the wells of a plate coated with streptavidin serving as a second solid support (SSS). After allowing reaction at 800 rpm at room temperature for 10 minutes, the bio-M-SSN-FSS complex was induced to be immobilized on the second solid support via the biotin serving as the second tag, so that an immobilized SSS-M-SSN-FSS complex was prepared.

[0153] Next, the liquid was carefully removed from the wells of the plate with a pipette, and the wells were washed 8 times with 300 .Math.L of a PB1 buffer supplemented with 25% propylene glycol (or 30% glycerol). The wells were washed 5 times with 300 .Math.L of a SB17T buffer, and finally the washing liquid was suctioned.

[0154] 100 .Math.L of a CAPSO elution buffer was added, and the immobilize SSS-M-SSN-FSS complex was shaken for 5 minutes so that the SS N-FSS complex was eluted. The SSN-FSS complex population was obtained by manually transferring 90 .Math.L of the solution from the wells of the plate to the wells of a PC R plate containing 10 .Math.L of a neutralization buffer.

[0155] For the SSN-FSS population thus obtained, the PCR was performed in the same manner as in Examples 1 and 2 to prepare double-stranded DNA. As described above, a biotin-conjugated forward primer and a reverse primer were used to prepare biotin-conjugated double-stranded DNA. The biotin-conjugated double-stranded DNA was immobilized on the first solid support (FSS), and the sense strand was eluted as described above, thereby finally obtaining a DNA aptamer library having ability to specifically bind to the protein molecule population in the sample and being immobilized on the first solid support (FSS).

[0156] A DNA aptamer library with higher specificity and affinity may be prepared by repeating the above processes on the prepared DNA library one or more times. However, since it is ideal that the DNA library specific to the protein molecule population of the sample reflects the diversity of protein molecules contained in the sample, the above processes may be performed only once, but the washing process is diversified and repeated to obtain a DNA aptamer library that reflects well the diversity of protein molecules in the sample.

<Example 4-2> Application to Differentiation Between Samples

[0157] The obtained DNA aptamer library immobilized on the first solid support (FSS) was used to distinguish six serum samples (analysis samples L5, L7, L8, L10, L13, and L14) derived from lung cancer patients and six serum samples (comparative samples N1 to N6) derived from normal persons.

[0158] As the samples, lung cancer patient serum samples and normal person serum samples provided by Seoul Asan Hospital (IRB approval number 2015-0607) were used. In the analysis, the same procedures as in Example 4-1 were performed on each sample to isolate DNA aptamer populations specifically binding to protein molecule populations in the test samples and the control samples in a state in which each of the aptamer populations was immobilized on the first solid support.

[0159] Specifically, the DNA aptamer library immobilized on the first solid support (FSS) was reacted with the sample to prepare an aptamer-protein complex population (C-SSN-FSS complex population). Next, a second tag was attached to the protein molecule of the complex, and then the complex was reacted in the wells of the plate coated with streptavidin serving as a second solid support, followed by washing. Next, the aptamer population coupled to the protein molecule population was eluted in a form coupled to the first solid support, i.e., an SSN-FSS complex form.

[0160] Using the eluted complex population as a template and using the primer suggested in Example 1, a collective double-stranded DNA fragment was obtained. An NGS library was prepared using Ion AmpliSeq Library Kit 20 (Ion Torrent, Thermo Fisher Scientific) according to the manufacturer's protocol. Next, the sequence of each DNA fragment was analyzed using Ion Torrent (104, Thermo Fisher), and the occurrence frequency of each DNA fragment (i.e., number or amount of DNA fragments) and an aptamer, which is comprehensive information on the occurrence frequency of each DNA fragment, were obtained.

[0161] The results are shown in FIGS. 6 to 10. FIGS. 6 and 7 show the number of types of binding aptamers, the total occurrence frequency (i.e., total number of binding aptamers), the average frequency, and the maximum frequency in each analysis sample (lung cancer sample) and in each comparative sample (non-lung cancer sample), respectively. FIGS. 8 and 9 show aptamer profiles for some aptamers (BioSign_serial number) in six lung cancer samples and aptamer profiles for some aptamers in normal samples, respectively. FIG. 10 shows comparison between aptamer profiles for some aptamers that differ in the occurrence frequency in the lung cancer samples and the normal samples.

[0162] Referring to FIGS. 6 to 10, it can be seen that the profile (the overall frequency of appearance of each DNA aptamer) of the DNA aptamer library reacted with the analysis target sample and the profile (the overall frequency of appearance of each DNA aptamer) of the DNA aptamer library reacted with the comparative sample are clearly distinguished.