Efficient target protein-specific ligand selection method using aptamer

Abstract

The present invention relates to: a method for efficiently selecting a high-affinity candidate ligand from a biological sample by using candidate ligands including a DNA barcode and an aptamer specifically binding to a target protein; and a kit therefor. Specifically, a target protein-aptamer-candidate ligand complex is formed by reacting a target protein, a candidate ligand, and an aptamer binding to the target protein, candidate ligands that do not form the complex are removed by nuclease treatment, and then a high-affinity candidate ligand is selected by primer extension and quantitative analysis of a DNA barcode of the candidate ligand bound to the complex. The method according to the present invention can ensure high specificity and reproducibility even in physiological environments such as cell lysates, and can be applied to various protein targets, and thus can be used in a wide range of bio-research fields such as new drug development and diagnostic ligand search.

Description

Efficient target protein-specific ligand screening method using aptamers

[0001] The present invention relates to an efficient method for selecting target protein-specific ligands using aptamers, and more specifically, to a method for efficiently selecting candidate ligands in a physiological environment using a candidate ligand comprising an aptamer that specifically binds to a target protein and a DNA barcode, and a kit for the same.

[0002]

[0003] DNA-Encoded Library (DEL) screening technology is widely used as a method that combines combinatorial chemistry and gene sequencing to rapidly discover candidate ligands that bind to target proteins from various compound libraries. However, conventional DEL screening methods have limitations in that they are primarily performed in a purified protein environment and thus fail to adequately reflect the complex physiological conditions in actual vivo. As a result, problems frequently occurred where candidate ligands selected under in vitro conditions exhibited low specificity or affinity in actual physiological environments.

[0004] In addition, conventional technology has limitations in securing high-affinity, high-specificity ligands because it is difficult to effectively eliminate the problem of false positives (background signals) caused by non-specific binding. Therefore, there is a need for the development of a new ligand selection platform that operates reproducibly even under physiological conditions, efficiently removes non-specific complexes, and simultaneously secures selectivity and affinity for target proteins.

[0005] The technical problem that the present invention aims to solve is to provide an efficient target protein-specific ligand screening method and screening kit using aptamers.

[0006] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0007] To achieve the above technical objective, one embodiment of the present invention provides a method for selecting a target protein-specific ligand, comprising the steps of: reacting a target protein, a candidate ligand, and an aptamer that binds to the target protein in a biological sample; and selecting a candidate ligand that forms a target protein-aptamer-candidate ligand complex through the reaction, wherein the candidate ligand comprises a DNA barcode and the aptamer comprises a DNA sequence capable of binding complementarily to the DNA barcode of the candidate ligand.

[0008] In an embodiment of the present invention, the biological sample may include a cell or a cell lysate.

[0009] In an embodiment of the present invention, the candidate ligand may be in the form of an amino acid, peptide, protein, or a small molecule covalently bonded to a DNA barcode.

[0010] In an embodiment of the present invention, the step of removing a candidate ligand that does not form the target protein-aptamer-candidate ligand complex may be further included.

[0011] In an embodiment of the present invention, the removal step may be performed by a nuclease.

[0012] In an embodiment of the present invention, the step of selecting the candidate ligand may be performed by forming an amplification product by extending the primer on the complex, and then performing a quantitative analysis of the amplification product.

[0013] In an embodiment of the present invention, the primer extension can be performed using nPfu DNA polymerase.

[0014] In an embodiment of the present invention, (a) an aptamer that binds to a target protein, (b) a candidate ligand comprising a DNA barcode, and (c) a nuclease, a primer, and a polymerase, wherein the aptamer may comprise a DNA sequence capable of binding complementarily to the DNA barcode of the candidate ligand.

[0015] The present invention relates to a method for screening target protein-specific ligands and provides a novel screening platform capable of screening candidate ligands that form target protein-aptamer-candidate ligand complexes even in cell lysates or environments simulating physiological conditions.

[0016] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0017] FIG. 1 relates to various selective approaches for DEL screening, comprising: (A) interaction-dependent PCR in cell lysate; (B) photo-crosslinking-based DEL screening on purified and immobilized proteins; (C) intracellular delivery via a DNA-templated affinity labeling strategy using cell-penetrating cyclic peptides; and (D) cell complex trap enrichment. ® (Cellular Binder Trap Enrichment ® , cBTE) (E) This relates to primer extension-mediated aptamer-gating DNA-encoding library screening (PEMAG-DEL) according to the present invention.

[0018] Figure 2 shows (A) the secondary structure of extended AptCA-II. (B) Positive control (PC): 4-sulfamoylbenzoic acid (CA II ligand) conjugated to three leucine residues. (C) Negative control (NC): Benzoic acid (non-target ligand) conjugated to three leucine residues. (D). Solution-phase verification: (Left) ΔC between PC and NC in RT-qPCR analysis. T =7 was observed, confirming target-specific enrichment. (Right) On 20% urea-denatured PAGE, PC showed a stabilized double-stranded band, while no band was observed for NC due to Exo I degradation. The faint band in the BSA control (Lane 4) reflects residual sulfonamide-BSA affinity. (E) HeLa lysate-based cytoplasmic screening: (Left) ΔC in RT-qPCR T =6 was maintained, proving robustness even in physiological environments. (Right) Strong PC bands were observed on the PAGE even in complex lysis environments, demonstrating the resistance of PEMAG-DEL to non-specific interference.

[0019] Figure 3 shows (A) ΔC depending on the presence or absence of CA II-specific aptamer (AptCA-II). T Value comparison. ΔC in the presence of AptCA-II T = 7(PC vs. NC), whereas ΔC in absence T = decreased to 3, demonstrating the importance of aptamers in targeted selective enrichment. (B) Degradation PAGE analysis of band intensity under NC and aptamer-free conditions showed negligible differentiation (similar band intensity) in the absence of AptCA-II, further confirming the necessity of aptamers for specificity. (C) The non-target control using an EGFR-specific aptamer (non-corresponding to CA-II) showed high C T Values ​​are shown to demonstrate that target recognition depends on aptamer-protein complementarity. (D) Highest ΔC between analyses with and without AptCA-II TA graph showing the difference, reinforcing the role of aptamers in improving screening resolution. (E) C relative to the orthogonal control group T Values; the positive control (PC) and the entire DEL library (125 members) show a significant shift.

[0020] Figure 4 shows (A) two ligands (a, b) selected for high similarity and one ligand (c) for low similarity selected through sequencing after screening the entire DEL library (125 members) under cell lysis conditions, the DEL library, and C of DEL (sulfonamide only without peptide). T Value comparison: a. Sulfo-His-His-Leu: C T = 19.96, b. Sulfo-His-His-Ala: C T = 19.60, c. Sulfo-Gly-Ala-Gly: C T = 24.96, d. DEL library: C T = 23.84, e. DEL (sulfonamide only without peptide): C T = 25.63 (B) Comparison of the above high-efficiency combinations under solution-phase conditions: a. Sulfo-His-His-Leu: C T = 18.38, b. Sulfo-His-His-Ala: C T = 20.40, c. Sulfo-Gly-Ala-Gly: C T = 25.96, d. Actual DEL: C T = 22.67, e. DEL (sulfonamide only without peptide): C T = 26.63.

[0021] Figure 5 relates to the analysis of CA II inhibition by selected sulfonamide-based peptides, comprising (A) the reaction mechanism in which CA II esterase activity is affected by the inhibitor (created in ChemDraw) and (B) the IC of the reference compound 4-sulfamoylbenzoic acid. 50= 13.82 μM Measurement Graph (C) IC50 of selected COOH-Ala-His-His-4-sulfamoylbenzoic acid 50 = 0.84 μM Measurement Graph (D) IC50 of selected COOH-Leu-His-His-4-sulfamoylbenzoic acid 50 = 0.98 μM The error bars (SD) in the measurement graph were calculated from two independent repeated data points in each experiment.

[0022] Figure 6 shows (A) identification of DNA-encoding library (DEL) members selected under strict conditions via 20% urea PAGE analysis. Distinct bands observed on the gel represent sequences enriched after selection, and the leftmost lane indicates molecular weight markers. Highlighted bands correspond to the amplification products of DEL members that showed high similarity in sequence alignment. The near absence of low-band signals indicates that non-binders were effectively removed, demonstrating the specificity and efficiency of this screening method. (B) identification of conjugation reaction products via 12% SDS-PAGE and Coomassie blue staining. Lane 1: Protein marker; Lane 2: CA II protein alone; Lane 3: Reaction with benzamide ligand (negative control); Lane 4: Protein-free condition (negative control); Lane 5: Reaction with sulfonamide ligand (positive control). (C) The final DEL product that formed a single band after all three amino acids were added was identified in 2% agarose gel electrophoresis, and was recovered through gel cutting and purification and used in PEMAG-DEL.

[0023] Figure 7 relates to the optimization of the exonuclease treatment reaction, wherein (A) time dependence was analyzed by varying the exonuclease (40 units) treatment time in positive / negative control groups to 45 minutes, 1.15 hours, 1.45 hours, and 2.15 hours. (B) Based on panel A data, positive (C under 1000-fold dilution conditionsT = 33) and voice (C T = 35) ΔC between control groups T = 2 observed. (C) Concentration dependence was evaluated by fixing the reaction time at 1.30 hours and varying the exonuclease concentration to 20, 40, 60, and 80 units. (D) Positive (C in Panel C data under the 1000-fold dilution condition T = 27) and voice (C T = 34) ΔC between control groups T = This indicates that 7 has been confirmed.

[0024] Figure 8 relates to the amplification curve of DEL screening using RT-qPCR, wherein (A) positive (C) when CA II protein and aptamer are present in solution. T =30.05) and voice (C T =36.87) Amplification curve of the control group. (B) Positive in the presence of BSA protein and CA II aptamer in solution (C T =36.78) and negative (C T =38.55) Control group. (C) Positive when CA II protein and aptamer are present in HeLa lysate (C T =29.90) and negative (C T =36.66) Control group. (D) Positive when BSA protein and CA II aptamer are present in HeLa lysate (C T =36.47) and negative (C T =36.88) Control group. (E) Positive when both CA II protein and aptamer are absent in HeLa lysate (C T =36.67) and negative (C T =37.97) Control group. (F) C of all experimental conditions T Graph integrating the values. The positive control group used 4-sulfamoyl benzoate-based DEL, and the negative control group used benzoate-based DEL.

[0025] Figure 9 is a representative image showing the color change of the reaction product as the inhibitor concentration increases in a 96-well plate, where the reference compound is 4-sulfamoylbenzoic acid and shows the inhibitory effects of Sulfo-HHL and Sulfo-HHA selected after DEL screening.

[0026] Figure 10 shows that (A) Tm=46 ℃ when CA II is alone, (B) Tm=54 ℃ when CA II + 4-sulfobenzoic acid is present, (C) Tm=59 ℃ when CA II + Sulfo-HHL is present, and (D) Tm=61 ℃ when CA II + Sulfo-HHA is present, indicating high binding strength.

[0027] Figure 11 shows the results of performing multiple sequence alignment with the ClustalW (EMBL) tool after purifying 125 types of DEL combinations using a kit column or gel cutting purification, and confirms that the HHA-sulfo and HHL-sulfo combinations are the most efficient combinations, exhibiting more than 90% sequence similarity.

[0028] Figure 12 relates to the expansion of PEMAG-DEL to PD-L1 immune checkpoint targets, comprising: (A) the chemical structure of a positive control (PC-P) in which the PD-L1 inhibitor BMS-8 is conjugated to a leucine triresin (Leu₃); (B) the chemical structure of a benzoic acid-Leu₃-based negative control (NC); and (C) comparative RT-qPCR amplification curves of a non-specific CA II aptamer (AptCA-II) and a PD-L1-specific aptamer (AptPD-L1). ΔC only in the presence of AptPD-L1. T A difference was confirmed, proving aptamer-mediated specificity. (D) 10% PAGE analysis under conditions using BSA instead of PD-L1. Confirmed that there were no non-specific signals, as both PC-P and NC showed almost no band intensity. (E) 10% urea PAGE analysis comparing the presence and absence of PD-L1 aptamers. Amplification products were identified only under conditions where aptamers were present. (F) PC-P showed ΔC compared to NC under PD-L1 spike conditions in HeLa lysate. TIt indicates =6.5, and a strong band was also confirmed on the page.

[0029] (G) ΔC of PD-L1 screening compared to the control group under non-aptamer, CA II aptamer, and BSA conditions T Value Summary Table. (H) ΔC depending on the presence or absence of PD-L1 aptamer T It is about value comparison graphs.

[0030] Fig. 13 shows (A) a positive (C) in the presence of PD-L1 protein and aptamer in HeLa lysis. T =28.30) and negative (C T =34.56) Control group. (B) Positive when BSA protein and PD-L1 aptamer are present in HeLa lysate (C T =33.97) and negative (C T =34.28) Control group. (C) Amplification curve under PD-L1 protein + peptide alone. (D) Amplification curve under PD-L1 protein + peptide and DNA barcode lacking both pharmacological functional groups.

[0031] Figure 14 relates to HPLC analysis data, specifically (A) Sulfo-His-His-Leu and (B) Sulfo-His-His-Ala.

[0032] Figure 15 relates to MS spectrum data, specifically (A) Sulfo-His-His-Leu and (B) Sulfo-His-His-Ala.

[0033] FIG. 16 relates to the NMR spectra of synthetic compounds, comprising: (A) 1H NMR spectrum of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)glycinate; (B) 1H NMR spectrum of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)alaninate; (C) 1H NMR spectrum of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)leucinate; (D) 1H NMR spectrum of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)tyrosinate; (E) 1H NMR spectrum of 2,5-dioxopyrrolidin-1-yl benzoate; and (F) 1H NMR spectrum of 2,5-dioxopyrrolidin-1-yl 4-sulfamoylbenzoate

[0034] Figure 17 shows fluorescence microscopy analysis (placeholder), where (A) a strong FAM fluorescence (green) signal localized to the cell surface by aptamer binding is observed in MDA-MB-231 cells treated with BMS-8 (PC). (B) almost no fluorescence signal is observed in cells treated with benzoic acid (NC). These representative images show that aptamers / DEL are retained in the cells under PC conditions, whereas they are not retained under NC conditions.

[0035] Figure 18 relates to the evaluation of aptamer concentrations in MDA-MB-231 cells through qPCR and gel analysis.

[0036] Figure 19 shows the results of qPCR and gel analysis according to aptamer culture time.

[0037] Figure 20 relates to qPCR and gel analysis under PC (BMS-8) and NC (Benzo) conditions in DEL screening targeting PD-L1.

[0038]

[0039] The present invention will be described in detail below.

[0040]

[0041] The present invention relates to a method for selecting target protein-specific ligands.

[0042] The method of the present invention comprises the steps of: reacting a target protein, a candidate ligand, and an aptamer that binds to the target protein in a biological sample; and selecting a candidate ligand that forms a target protein-aptamer-candidate ligand complex through the reaction, wherein the candidate ligand comprises a DNA barcode, and the aptamer comprises a DNA sequence capable of binding complementarily to the DNA barcode of the candidate ligand.

[0043] In the present invention, the target protein refers to a protein to which a candidate ligand binds, thereby exhibiting or inhibiting biological activity. In the present invention, the target protein mediates the binding of candidate ligands within a spatially limited region through specific binding with an aptamer, serving as a key target for candidate ligand selection. Carbonic Anhydrase II (CA II), which is representatively used in the present invention, is an enzyme protein associated with the regulation of the tumor microenvironment and ophthalmic diseases. Programmed Death Ligand-1 (PD-L1), an immune checkpoint protein used in another embodiment of the present invention, plays a crucial role in anticancer immunomodulation, and the applicability of the method of the present invention has been experimentally demonstrated. The target protein of the present invention can be extended to other therapeutic target proteins, such as antibodies, receptor proteins, and intracellular signaling proteins.

[0044] The binding of the aforementioned target protein to candidate ligands is spatially restricted through aptamers, thereby minimizing the background of non-specific binding. Through the aptamers of the present invention, stable complexes can be formed even in various physiological environments (including cell lysates), enabling the discovery of candidate ligands under conditions similar to actual biological environments. By designing and applying appropriate aptamer sequences according to the type of target protein, the present invention can be extended to various application fields such as anticancer, immunomodulation, and enzyme inhibition.

[0045] The above biological sample may include cells or cell lysates. The biological sample used in the present invention refers to a biologically derived material in which a target protein may be present or expressed, and includes not only simply purified protein solutions but also cells or complex matrices derived from cells. Since such biological samples can reflect the structural stability, folding, or interaction with cofactors of the target protein in a physiological state, they are utilized as an environment for evaluating the binding specificity and physiological fidelity of a ligand.

[0046] Examples of biological samples include cells, cell lysates, or cell fraction samples. Cell samples include cultured animal cells (e.g., HeLa, MDA-MB-231, etc.), microbial cells, or genetically modified cells, and allow for the evaluation of ligand binding when the target protein is expressed on the cell membrane or present within the cell. Cell lysates are solutions obtained by physically or chemically lysing cells, mimicking a complex biological environment where intracellular proteins, nucleic acids, and metabolites are mixed. The advantage of using such cell lysates is that ligand selectivity can be verified even in environments of non-specific or competitive binding. Additionally, cell fraction samples are samples containing specific fractions of a cell (e.g., cytoplasm, nucleus, membrane fraction, etc.) and can be prepared to match the cellular compartment where the target protein is predominantly located.

[0047] The target protein-specific ligand screening method according to the present invention can directly and quantitatively detect whether a target protein and a ligand bind by utilizing the biological sample. In particular, screening performed in a cell lysate environment reflects physiological conditions more faithfully than experiments based on purified proteins, and is therefore advantageous for evaluating the binding potential and efficacy of the ligand in actual vivo conditions.

[0048] In addition, the method of the present invention is characterized by being able to effectively select ligands that specifically bind to a target protein through an accuracy filtering mechanism based on aptamer-mediated selectivity and primer extension, despite interference caused by proteases, nucleases, non-specific binding proteins, etc., present in the cell or cell lysate. The candidate ligand may be in the form of a DNA barcode covalently bonded to an amino acid, peptide, protein, or small molecule. The candidate ligand is finally selected during the screening process based on binding affinity and specificity by selectively binding during the formation of the aptamer-target protein complex.

[0049] Amino acid-based candidate ligands can utilize various amino acid derivatives, such as leucine, alanine, histidine, glycine, and tyrosine, as their basic backbones. Peptide-based candidate ligands utilize peptide sequences in which multiple amino acids are linked in a linear or cyclic manner, which increase the potential for interaction with the active site of a target protein. Small molecule-based candidate ligands include derivatives of small molecule compounds such as sulfonamides and benzoic acid, and can utilize functional groups previously known as target protein inhibitors. Protein-based candidate ligands may also include ligands in the form of small proteins or protein fragments containing specific binding sequences.

[0050] All candidate ligands are linked to unique DNA barcode sequences via covalent bonds, allowing for the quantitative evaluation of binding affinity through RT-qPCR analysis following the screening process. A library of tens to hundreds of candidate ligands can be rapidly constructed using a combinatorial approach (split-pool synthesis). The combination of the chemical diversity of candidate ligands and DNA barcode-based high-speed analysis capabilities enables the screening of hundreds of ligand affinities within a single experiment. Non-specific binding backgrounds are removed through nuclease treatment and aptamer-based gating, ensuring that only high-affinity, high-specificity ligands are ultimately selected.

[0051] The above aptamer refers to a single-stranded DNA or RNA oligonucleotide sequence synthesized to bind to a specific target protein with high specificity and affinity. Similar to antibodies, aptamers selectively recognize specific binding sites on target proteins, and because they have a small molecular weight and are chemically stable, they can be used repeatedly under various biochemical conditions. In the present invention, aptamers are utilized as a key component mediating the selective binding between a candidate ligand and a target protein.

[0052] DNA aptamers are based on DNA sequences, and the carbonic anhydrase II (CA II)-specific DNA aptamers and PD-L1-specific DNA aptamers primarily used in this invention fall into this category. RNA aptamers are based on RNA sequences and can exhibit high-affinity binding characteristics to protein targets. They can be used instead of DNA aptamers if necessary. As modified aptamers, aptamers in which chemical modifications are introduced to nucleotide ends or bases to improve stability, binding affinity, and half-life in vivo can also be applied to this invention.

[0053] Aptamers exhibit high binding specificity and strong affinity for target proteins, inducing candidate ligands to bind exclusively to the target protein. Furthermore, their sequences are designed to enable complementary binding to DNA barcodes, allowing for control over complex formation at the molecular level. By maintaining structural stability even in complex physiological environments such as cell lysates, they contribute to the reduction of false positives. Compared to antibody-based technologies, they offer lower production costs and easier synthesis, enabling high-speed screening and customized application to various target proteins.

[0054] In the present invention, the step of reacting a target protein, a candidate ligand, and an aptamer that binds to the target protein refers to the process of mixing the target protein, an aptamer that specifically binds thereto, and a candidate ligand containing a DNA barcode to form an aptamer-target protein-candidate ligand complex. This step is the most critical step in the ligand selection process, enabling the binding between the target protein and the candidate ligand to occur specifically and selectively through aptamer mediation.

[0055] The reaction according to the present invention first mixes a target protein and an aptamer to form a stable protein-aptamer complex, and then adds a candidate ligand containing a DNA barcode to finally form a target protein-aptamer-candidate ligand complex. In this process, the aptamer acts as a molecular gating agent to mediate the binding between the target protein and the candidate ligand, thereby minimizing the non-specific binding of the candidate ligand to the target protein. The reaction can be carried out in complex environments such as physiological buffers or cell lysates, and the efficiency of complex formation is maximized under optimal pH, ion concentration, and reaction time conditions.

[0056] The aptamer according to the present invention specifically binds to the target protein binding site, thereby providing a binding surface that allows candidate ligands to selectively interact with the target protein. Furthermore, it is applicable to physiological conditions, and stable complex formation occurs even under conditions similar to actual biological environments, such as cell lysates, minimizing false positives. Only the complex formed at this stage is selectively maintained even after nuclease treatment, leading directly to quantitative evaluation through primer extension and RT-qPCR analysis.

[0057] In an embodiment of the present invention, the method may further include a step of removing candidate ligands that do not form the target protein-aptamer-candidate ligand complex. This step refers to a process of selectively degrading and removing candidate ligands that do not specifically bind to the target protein and aptamer, i.e., non-specific complexes or unbound free candidate ligands, thereby minimizing false positives (background signals) during the analysis process.

[0058] In this step, a nuclease, preferably Exonuclease I, is treated to the reaction mixture in which the target protein-aptamer-candidate ligand complex has been formed. Exonuclease I acts specifically on single-stranded DNA (ssDNA) to eliminate non-specific signals by degrading the DNA barcode of the candidate ligand that is not bound to the complex. On the other hand, the DNA barcode protected within the complex is preserved without being cleaved because enzymatic access is blocked. After enzymatic treatment, the reaction is inactivated at an appropriate temperature to avoid affecting subsequent primer extension and amplification steps.

[0059] At this stage, selectively cleaving only the DNA barcodes of unbound candidate ligands significantly enhances the sensitivity and specificity of screening. Furthermore, candidate ligands bound within the complex are stably preserved as access by degrading enzymes is physically blocked. By suppressing false positives through a single-strand elimination step that does not form a complex using nucleases, it is possible to accurately evaluate only candidate ligands that form target protein-aptamer-candidate ligand complexes in primer extension reactions and RT-qPCR-based quantitative analysis. In addition to Exonuclease I, other single-strand-specific nucleases can be used, and the method is equally applicable regardless of the type of target protein or aptamer.

[0060] In an embodiment of the present invention, the method may further include the step of forming an amplifiable product by performing primer extension in the complex. This refers to a process of selecting high-affinity candidate ligands by removing candidate ligands that did not form a target protein-aptamer-candidate ligand complex, converting the DNA barcode sequence of a candidate ligand bound to the complex into a double-stranded DNA form through a primer extension reaction, and quantitatively analyzing the result.

[0061] For the target protein-aptamer-candidate ligand complex remaining after the deconjugation step, primers that bind complementarily to the candidate ligand's DNA barcode sequence can be provided, and a primer extension reaction can be performed using nPfu DNA polymerase. Since the double-stranded DNA formed at this stage selectively reflects only the candidate ligand bound to the complex, differences based on binding affinity manifest as the amount of amplification product. Subsequently, the C of the amplification product is analyzed via RT-qPCR. T By measuring the value, the binding strength of each candidate ligand is quantitatively evaluated, and C T Select the candidate ligand with the lowest value.

[0062] At this stage, primer extension is performed only on the DNA barcode bound to the complex, enabling highly specific analysis with the non-specific binding background eliminated. Additionally, RT-qPCR analysis quantifies the binding affinity of each candidate ligand using C T By providing values, the relative binding strength between candidate ligands can be objectively compared. Through the primer extension reaction, even trace amounts of complex binding signals can be amplified and detected, enabling high-sensitivity screening. At this stage, candidate ligands selected for higher ΔCT values ​​compared to unbound ligands can be easily advanced to additional chemical synthesis, biochemical activity evaluation, and pharmacological efficacy verification steps.

[0063] The present invention relates to a kit for screening target protein-specific ligands. The kit comprises an aptamer that binds to a target protein, a candidate ligand containing a DNA barcode, a nuclease, a primer, and a polymerase. It refers to a product provided to enable the convenient execution of the target protein-specific ligand screening method according to the present invention in a single package form. This kit can be designed to allow a user to rapidly discover candidate ligands that bind to a target protein without additional reagent preparation or complex optimization processes.

[0064] The aptamer included in the kit according to the present invention may be a DNA or RNA aptamer capable of specifically binding to a target protein and forming a sequence complementary to a candidate ligand. The candidate ligand library containing a DNA barcode may be in the form of various compounds, such as amino acids, peptides, and small molecules, covalently bonded to the DNA barcode. The nuclease may be an enzyme that removes the DNA barcode of an unbound candidate ligand, such as Exonuclease I. Primers and nPfu DNA polymerase are included as reagents required for primer extension and amplification reactions.

[0065] The kit supports the entire process, from the formation of target protein-aptamer-candidate ligand complexes to the removal of unconjugated molecules, primer extension, amplification, and quantitative analysis, within a single package. It is versatile enough to be applied to various target proteins and allows for the design of customized libraries according to research objectives. Users can rapidly and accurately discover high-affinity candidate ligands without the need for complex equipment or additional reagent preparation. It is advantageous for experimental standardization, providing highly reproducible results and enabling easy extension to subsequent validation studies.

[0066]

[0067] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0068]

[0069] I. Primer Extension-Mediated Aptamer-Gated DNA-Encoded Library Screening for Physiologically Relevant Ligand Discovery

[0070] Experimental method

[0071] 1. Prepare the DNA-encoded library (DEL)

[0072] DNA encoding libraries (DELs) were constructed through sequential conjugation, deprotection, ligation, and duplex formation steps. Specifically, five amino acids (15 mM, 3 μL each) were individually conjugated to SM-DNA O1-O5 (100 μM, 12 μL), followed by deprotection and purification using 10% piperidine. This process was repeated through additional amino acid conjugation, duplex formation using appropriate splint DNA and code-DNA, and ligation using T4 DNA ligase (12,000 units, 16 °C, overnight) in 1× ligation buffer until sulfonamide was finally introduced. The reaction was performed in a total volume of 20 μL using 100 mM phosphate buffer at pH 8.5, piperidine was mixed with DMSO in a 1:1 ratio, and DNA was eluted in 15 μL. Detailed descriptions are provided in the supplement information of the paper J Med Chem. 2025 Aug 14;68(15):16227-16236. All compounds were purified using gel cut purification, and the main compounds were further purified using analytical HPLC (C18 column, 220 nm UV detection) to ensure a purity of over 95%.

[0073] Gel electrophoresis was performed on a 20% modified polyacrylamide gel (PAGE). A 20% non-modified polyacrylamide gel solution (total 5 mL) was prepared by mixing 40% Acrylamide / Bis solution (2.5 mL), 10× TBE buffer (0.5 mL), urea (Urea, 8578-4400, DAEJUNG; 2.5 g), 20% ammonium persulfate solution (dissolved in distilled water; 20 μL), and tetramethylethylenediamine (TEMED, Sigma-Aldrich, USA; 5 μL). 4-Sulfobenzoic acid, 98% (CAS 636-78-2), benzoic acid (CAS 99-06-9), and BMS-8 [1-(3-Bromo-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic acid] (CAS 1675201-90-7) were purchased from BLD Pharm (China). DEL products containing the library were purified by gel cutting on a 2% agarose gel (Thermo Scientific™ TopVision Agarose) and then processed using AccuPrep from Bioneer (Korea). ® The gel was purified using a PCR / Gel Purification Kit. The gel was prepared using a BIO-RAD Mini-PROTEAN Tetra Cell (USA) instrument and developed at 80 V for 6 hours. After development, the gel was stained with EtBr solution for 5 minutes, washed with water for 2 minutes, and then imaged using a transiluminator.

[0074] A total of 27 oligonucleotides used for this study are listed in Table S1 of the supplementary materials of J Med Chem. 2025 Aug 14;68(15):16227-16236.

[0075]

[0076] 2. Primer Extension-Mediated Aptamer-Gated DNA-Encoded Library Screening (PEMAG-DEL)

[0077] To conjugate the CA-II aptamer to the CA-II protein, the aptamer was first heated at 95 °C for 5 minutes and then slowly cooled to room temperature to form a secondary structure. The formed aptamer (100 μM, 3 μL) was mixed with CA-II protein (8.33 μM, 4 μL; approx. 1 μg), HEPES buffer (20 μM, 1 μL), MgCl₂ (10 mM, 1 μL), NaCl (1 M, 1 μL), and sterile water to adjust the final volume to 10 μL. To simulate the cytoplasmic environment, HeLa cell lysate was added at a concentration of 10% of the total reaction volume, and the mixture was incubated overnight at 4 °C. Subsequently, DEL compound (~100 μM) was added and cultured at room temperature for 1 hour, followed by at 4 °C for 1 hour to induce stable double-strand formation between the CA-II aptamer-CA-II protein and the DEL complementary sequence.

[0078] Preparation of DEL: DNA-encoding libraries (DELs) were prepared using a multi-step process. First, five amino acids (leucine, alanine, histidine, glycine, and tyrosine; 15 mM, 3 μL each) were added to SM-DNA 01 / 02 / 03 / 04 / 05 (100 μM, 12 μL each) in separate tubes (5 tubes). Subsequently, a deprotection reaction was performed with 10% piperidine, and after purification using a kit column, the samples were pooled and divided into 5 tubes. In the second step, the same five amino acids (15 mM, 3 μL each) were added again, and double strands were formed using Splint DNA-1 (100 μM, 11 μL) and code-DNA-11 / 12 / 13 / 14 / 15 (100 μM, 11 μL each), followed by a primary ligation reaction. Next, purification, pooling, and dispensing were performed following the Fmoc deprotection reaction (10% piperidine). Thirdly, five types of amino acids (15 mM, 3 μL each) were added, and a double strand was formed using Splint DNA-2 (100 μM, 10 μL) and code-DNA-21 / 22 / 23 / 24 / 25 (100 μM, 10 μL each), followed by a secondary ligation reaction. Subsequently, the Fmoc deprotection reaction, purification, pooling, and dispensing were repeated. Next, sulfonamide (11 mM, 3 μL) and benzamide (11 mM, 3 μL) were added, and a double strand was formed using Splint DNA-3 (100 μM, 9 μL) and code-DNA-36 (positive control) / code-DNA-37 (negative control; 100 μM, 9 μL each). Finally, a tertiary linkage reaction was performed, and the product was purified with a gel cutting tablet and eluted in sterile water.

[0079] Amino acid and sulfonamide addition reactions were always performed in 100 mM phosphate buffer (pH 8.5), and the total reaction volume was adjusted to 20 μL. Piperidine was pre-mixed with DMSO in a 1:1 ratio. The ligation reaction was carried out at 16 °C for approximately 16 hours using 1× T4 DNA ligase buffer (50 mM Tris-HCl, pH 7.5; 10 mM MgCl₂; 10 mM DTT; 1 mM ATP) and 12,000 units of T4 DNA ligase. Double strand formation was performed by heating at 95 °C for 5 minutes followed by slow cooling to room temperature. The final DEL product was purified by gel cleavage (Figure 6).

[0080] DEL Screening Optimization: The first amino acid (Fmoc-protected leucine / alanine / histidine / glycine / tyrosine-NHS ester) was conjugated to the 3' terminal C6 amine (modification site) of SM-DNA-01 / 02 / 03 / 04 / 05, followed by the Fmoc deprotection reaction. Subsequently, the deprotected first amino acid formed an amide bond with the second amino acid (Fmoc-protected leucine / alanine / histidine / glycine / tyrosine-NHS ester), and this process was repeated sequentially until the introduction of sulfonamide. At each amino acid addition step, a unique 6-base DNA barcode corresponding to the amino acid was conjugated to the preceding code via a linkage reaction to construct an accumulated encoding sequence (Figures 6A, B).

[0081] Aptamer-CAII Protein-DEL Complex Formation: Based on prior research, various buffer compositions were tested to conjugate the aptamer to the target CA-II protein. Evaluation results showed that the combination of 2 μM HEPES, 1 mM MgCl₂, and 100 mM NaCl was most suitable for conjugation. During the reaction, the aptamer:protein ratio was maintained constant at 10:1. Approximately 3.33 μM of CA-II protein and 30 μM of CA-II aptamer were used under a total volume of 10 μL for a conjugation reaction of about 16 hours at 4 °C. Subsequently, 6.6 μM of DEL was added to bring the total reaction volume to 15 μL. Since exposure to high temperatures can inhibit aptamer-protein binding, the double-strand formation of the protein-aptamer conjugate and DEL was carried out at room temperature. This approach allows the complementary region of DEL to efficiently hybridize with the extended complementary region of the aptamer while maintaining the stability of the conjugate.

[0082] 3. Exonuclease treatment and primer extension

[0083] The cultured reaction mixtures underwent an Exonuclease-I (Exo-I) treatment process. For each reaction, 1× Exonuclease-I buffer [67 mM Glycine-KOH (pH 9.5), 6.7 mM MgCl₂, 10 mM 2-mercaptoethanol] and Exonuclease-I enzyme (20 units) were added, followed by incubation at 37 °C for 1 hour and 30 minutes, and then heat inactivation at 80 °C for 20 minutes. Subsequently, primer extension was performed at 37 °C for 2 hours using a mixture of nPfu-special polymerase (2 units), 1× nPfu-special A buffer, and 1 mM dNTPs. A ligation step was then added to seal the junction between the extended region and the splint DNA. The final product was purified by gel cutting, and its purity was evaluated using a NanoDrop spectrophotometer. The purified product was subsequently used as a PCR template. Real-time qPCR was performed according to the manufacturer's instructions using TOPreal™SYBR Green qPCR High-ROX PreMIX with 1 μM each of the designed forward and reverse primers. Amplification conditions included initial denaturation at 95 °C for 12 minutes, followed by a total of 40 cycles consisting of 95 °C for 10 seconds, 45 °C for 15 seconds, and 72 °C for 30 seconds, and included melt curve analysis. Real-time qPCR data were analyzed using Design and Analysis Software version 2.8.0.

[0084] Exonuclease Treatment Optimization: The specificity of this method depends on the 3'→5' single-strand DNA cleavage activity of Exonuclease I. When a selective aptamer is conjugated with CA II, the sulfonamide-labeled DEL forms an extended overhang of the aptamer and a stable double strand, occupying the 3' end of DEL, and the 3' end of the aptamer also double-strands 12 bp complementary to DEL. Consequently, Exonuclease I selectively cleaves only the unreacted DEL / aptamer single strands / 3' ends. However, since excessive enzyme concentration or treatment time can negatively affect the main reaction, optimization was performed using enzyme concentration (20-80 units) and time (30 minutes-2.5 hours) as variables. Based on the experimental results, it was confirmed that the conditions of 20 units and 1.5 hours were optimal (Figure 7).

[0085] Primer Extension: Primer extension proceeds only if the CA II-aptamer-DEL conjugate remains after Exonuclease I treatment (positive reaction); if no conjugate remains (negative reaction), primer extension does not occur. This step is a key process for verifying the operation of this screening concept, and targets can be detected through subsequent PCR amplification even if reaction efficiency is very low due to exonuclease cleavage.

[0086]

[0087] 4. Rigorous DEL Screening Conditions for Optimal Ligand Identification

[0088] To select the most efficient combination among the 125 DEL combinations via qPCR amplification, each reaction sample was diluted 1000-fold, and the target and primer concentrations were minimized by limiting the primer concentration to 50 nM per 20 μL reaction. This established conditions where only the most efficient combination among the 125 DEL combinations could be amplified by qPCR. Subsequently, C TOnly products with a value of 35 or higher were selected and purified, and Sanger sequencing was performed. Sequencing data were analyzed using CLUSTALW, and the most efficient combination was identified based on sequence similarity with 125 DEL libraries.

[0089]

[0090] 5. CA-II binding assay

[0091] To verify the efficacy of ligands selected via PEMAG-DEL, the combinations exhibiting the highest concentrations—Sulfo-His-His-Leu, Sulfo-His-His-Ala, and the parent compound (4-sulfamoylbenzoic acid)—were commercially synthesized, and their binding affinities were evaluated via carbonic anhydrase II (CA II) enzyme inhibition assays. The esterase activity of CA II, which is well-established for sulfonamide binding, was quantified by monitoring the hydrolysis of 4-nitrophenyl acetate to 4-nitrophenol (λmax = 408 nm) in 50 mM HEPES buffer (pH 7.2). In a 96-well plate, CA II (1 μM) was reacted with inhibitors diluted to various concentrations (1 mM–1 nM) at 25 °C for 15 minutes, after which the substrate (final concentration 0.45 mM) was added. Initial reaction rates were measured using a spectrophotometer, and the IC50 values ​​were calculated. 50 The values ​​were calculated using non-linear regression analysis (log[inhibitor] vs. normalized response, variable slope) in GraphPad Prism 8 software.

[0092]

[0093] 6. Protein stability analysis using Thermal Shift Assay (TSA)

[0094] Binding interactions between recombinant CA-II protein and sulfonamide derivatives selected via PEMAG-DEL screening were evaluated. This analysis monitored protein denaturation upon ligand binding using a fluorescent dye (SYPRO™ Orange). CA-II protein was diluted in sterile water to an optimal concentration, and standard 4-sulfamoylbenzoic acid inhibitors, Sulfo-HHA, and Sulfo-HHL were diluted in DMSO and added in specific molar ratios. Samples were prepared in 96-well PCR plates, and fluorescence signals were monitored using a real-time qPCR instrument while increasing the temperature from 25 °C to 90 °C at a rate of 1 °C / min. The melting temperature (Tm) of each sample was determined from the first derivative of the melting curve.

[0095]

[0096] 7. Verification of Universality of PEMAG-DEL Targeting PD-L1

[0097] To apply PEMAG-DEL to PD-L1, the PD-L1 aptamer (100 μM) was first heated at 95 °C for 5 minutes and then cooled to room temperature to form a folded structure. The formed aptamer was composed of recombinant PD-L1 (8.33 μM), DPBS (Ca 2+ , Mg 2+The mixture was mixed with (including), BSA (1 mg / mL), and sterile water to a final volume of 10 μL, and incubated overnight at 4 °C to form aptamer-protein complexes. Subsequently, 1 μL of DEL conjugate (PC-P and NC) was added, and the mixture was incubated at room temperature for 2 hours and at 4 °C for 1 hour; in some reactions, an equal volume of buffer was replaced with 10% (v / v) HeLa cell lysate. Exonuclease-I treatment was performed at 37 °C for 90 minutes under conditions of 1× Exo-I buffer and 20 U Exo-I, followed by heat inactivation at 80 °C for 20 minutes. Primer extension was performed at 37 °C for 2 hours using 1× nPfu A buffer, 1 mM dNTP, and nPfu-special polymerase (2 U), and the ligation sites were subsequently sealed using T4 DNA ligase (12,000 units, 1× ligase buffer, 16 °C, overnight). The extension products were purified by gel cutting and quantified using NanoDrop. Real-time qPCR was performed in StepOne Software v2.3, including initial denaturation at 95 °C for 12 minutes, 40 cycles under conditions of 95 °C for 10 seconds, 45 °C for 15 seconds, and 72 °C for 30 seconds, and melting curve analysis.

[0098] Validation targeting PD-L1: After establishing the performance against CA II (Figure 2), we evaluated whether this aptamer-gating primer extension system could be generalized to clinically important targets. The immune checkpoint protein PD-L1 was selected, as it is known that PD-L1 inhibition reactivates cytotoxic T cells by blocking PD-1 / PD-L1 signaling. A reported PD-L1-specific DNA aptamer (nanomolar affinity) (Reference 36) was used as the gating element. As the small molecule ligand, the known PD-L1 inhibitor BMS-8, which possesses an amine functional group suitable for NHS conjugation, was selected. For the evaluation of the assay, BMS-8-Leu₃ (positive control, PC-P) and benzoic acid-Leu₃ (negative control, NC; no PD-L1 affinity) were synthesized (Figure 12A, B). Replacing the PD-L1 aptamer with a non-targeted CA II aptamer minimized the discriminative power (ΔC T Degradation) Aptamer selectivity was emphasized (Figure 12C). In addition, ΔC in screening using BSA as a target substitute T = 1.0 was observed, confirming that the observed signal depends on PD-L1 binding (Fig. 12D). Subsequently, as a result of performing PEMAG-DEL under the same conditions as CA II, the ΔC between PC and NC in the HeLa solution T By confirming a distinct difference of = 6.5, it was demonstrated that effective aptamer-gating discrimination is possible under both conditions (Fig. 12F). ΔC obtained under various control conditions, such as non-aptamer, uncompensated CA II aptamer, and off-target BSA. T As a result of the comparison, PC-P / PD-L1 consistently had the lowest C T It indicated the specific enrichment of target-dependent binding (Fig. 12G). Additionally, removing the aptamer resulted in ΔC Tg decreased to 2.8, suggesting background primer extension in the absence of gating (Fig. 12E, H). In other negative controls as well, high C was observed under conditions where only the peptide portion was present (Fig. 12C) or under DNA barcode conditions lacking both the peptide and pharmacological functional groups (Fig. 13D). T ...was observed, further supporting the specificity of the method. These results demonstrate that PEMAG-DEL can be successfully extended to unrelated protein targets using independently developed aptamers, supporting its utility as a universal ligand discovery platform.

[0099] 8. Preparation of Synthetic Compounds

[0100] Synthesis of 2,5-Dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)glycinate

[0101] Fmoc-Gly-OH and N-hydroxysuccinimide were dissolved in anhydrous methylene chloride under an inert atmosphere, and the solution was cooled to 0 °C. Dicyclohexylcarbodiimide was spotted to activate the acid, and the mixture was stirred at room temperature for 12–16 hours. Dicyclohexylurea was removed from the resulting mixture by filtration, and the filtrate was concentrated and purified by recrystallization (or chromatography) to obtain the active ester. The structure of the product was confirmed by characteristic ¹H NMR signals, such as Fmoc aromatic protons and succinimidyl methylene resonance.

[0102] Synthesis of 2,5-Dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)alaninate

[0103] The same protocol as the synthesis of the glycine derivative above was applied, but Fmoc-Gly-OH was replaced with Fmoc-Ala-OH.

[0104] Synthesis of 2,5-Dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)leucinate

[0105] The same protocol as the synthesis of the glycine derivative above was applied, but Fmoc-Gly-OH was replaced with Fmoc-Leu-OH.

[0106] Synthesis of 2,5-Dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)tyrosinate

[0107] The same protocol as the synthesis of the glycine derivative above was applied, but Fmoc-Gly-OH was replaced with Fmoc-Tyr-OH.

[0108] Synthesis of 2,5-Dioxopyrrolidin-1-yl benzoate

[0109] The same protocol as the synthesis of the glycine derivative above was applied, but Fmoc-Gly-OH was replaced with Fmoc-Benzoate-OH.

[0110] Synthesis of 2,5-Dioxopyrrolidin-1-yl 4-sulfamoylbenzoate

[0111] The same protocol as the synthesis of the glycine derivative above was applied, but Fmoc-Gly-OH was replaced with Fmoc-1-yl 4-sulfamoylbenzoate-OH.

[0112] Synthesis of Sulfo-HHL and Sulfo-HHA

[0113] The final peptide conjugates, Sulfo-His-His-Leu and Sulfo-His-His-Ala, were custom-synthesized by solid-phase peptide synthesis at Peptron Co., Ltd. (Daejeon, Korea) (Figs. 14, 15).

[0114]

[0115] result

[0116] 1. Research Design and DEL Establishment

[0117] To validate the PEMAG-DEL concept, 4-sulfamoylbenzoic acid (IC50-15μM), a well-known CA II ligand, was selected as the core scaffold. To increase screening efficiency and discover ligands with enhanced affinity, a combinatorial library was designed in which five unique amino acids—leucine, alanine, histidine, glycine, and tyrosine—were each linked to a unique 6-base sequence DNA barcode. This design generated a library of 125 unique ligands, with each ligand conjugated to 4-sulfamoylbenzoic acid. To establish the robustness of the method, two control compounds were synthesized: a positive control (PC) in which three leucine residues are attached to 4-sulfamoylbenzoic acid (4-sulfamoylbenzoic acid-Leu-Leu-Leu, Fig. 2B), and a negative control (NC) in which three leucine residues are attached to benzoic acid (benzoic acid-Leu-Leu-Leu, Fig. 2C, Fig. 6).

[0118] 2. Screening Phase and Verification

[0119] General screening began with a step to ensure proper secondary structure formation by cooling AptCA-II from 95 °C to 25 °C to refold it, followed by incubation with CA II in 1X binding buffer overnight at 4 °C to promote target binding. According to the hypothesis, positive controls (PC) and negative controls (NC) were added to the solution at 25 °C for 2 hours and then incubated at 4 °C for 1 hour; this allowed PC to form a stable double strand with the aptamer's toehold domain, while NC bound to the same domain but lacked affinity for CA II, resulting in an unstable double strand that was more easily exposed to Exo-I digestion. To eliminate non-specific interactions, the mixture was treated with Exonuclease-I (Exo I, 20 units) at 37 °C for 1.5 hours, followed by enzyme inactivation at 80 °C for 20 minutes. This step selectively degraded unhybridized SM-DNA to enrich the target binding complex (Figure 1E).

[0120] Next, primer extension was performed at 37°C for 2 hours using a mixture of nPfu-special polymerase (2 units), 1X nPfu-special A buffer, and 1 mM dNTPs; this process stabilized the double-stranded structure by extending the SM-DNA strands according to the aptamer template. During the primer extension step, the splint DNA hybridized to the code DNA intermittently became unstable, leading to fragmentation of the aptamer sequence. To address this, the extended product was further treated with T4 DNA ligase overnight at 16°C; this additional ligation step effectively sealed the junctions between DNA fragments, ensuring a continuous and covalently linked product. The final solution was analyzed by RT-qPCR using forward primers complementary to the 5′ end of the aptamer and reverse primers complementary to the SM-DNA barcode. ΔC between positive controls (PC) and negative controls (NC) T The value was 7, confirming significant enrichment of the target binding complex (Figure 2D left). In addition, urea PAGE analysis showed a distinct difference in band intensity between PC and NC, further verifying the selectivity of this method (Figure 2D right, lanes 3 and 5).

[0121] 3. Physiological verification in cell lysate

[0122] To evaluate the physiological accuracy of PEMAG-DEL, screening was performed in HeLa cell lysates, a complex matrix that replicates intracellular congestion and competitive binding composed of soluble proteins, nucleic acids, and metabolites. By adding cell lysates at a concentration of 10% (v / v) of the total reaction volume, analytical compatibility was ensured while maintaining physiologically appropriate concentrations of non-target biomolecules (total protein approximately 3–5 mg / mL). Notably, even under these conditions, ΔC between the positive control (PC) and the negative control (NC)T The value remained at 6, which corresponds to the solution phase condition (ΔC T It was confirmed that this method possesses strong resistance to macromolecular interference, as the performance observed in = 7) was similar (Figure 2E, left). This consistency highlights that PEMAG-DEL can induce target-selective hybridization despite the presence of abundant components in the cell lysate, such as nucleases, proteases, and non-specific DNA-binding proteins. Important validation was performed via urea PAGE analysis, which revealed a distinct difference in band intensity between PC and NC in the cell lysate environment. While the signal of NC was minimized due to Exo I-mediated degradation of unbound ligands, PC maintained a strong band corresponding to the stabilized SM-DNA:AptCA-II double strand (Figure 2E, right). This cross-validation demonstrates that PEMAG-DEL effectively minimizes the multipharmacological noise commonly problematic in cell lysate-based DEL screening by combining aptamer-induced spatial selectivity and enzymatic accuracy filtering.

[0123] 4. Specificity Verification Experiment of PEMAG-DEL

[0124] To rigorously verify the specificity of PEMAG-DEL, we performed a series of negative control experiments under the same screening conditions using the non-target protein BSA in both solution-phase and cell lysate conditions. As expected, in the presence of BSA in both cell lysate and solution-phase conditions, PC(C T -38) and NC(C T ΔC between -39) TNo significant difference in values ​​was observed (Figures 8B and 8D, right side of Figure 2E, lanes 2 and 4). This confirmed that the aptamer selectively induces DEL hybridization only in the presence of the target protein (CA II). This result emphasizes that the present method relies on specific recognition between the aptamer and the protein rather than non-specific interactions. Additionally, further verification was performed through three additional control experiments: (1) SM-DNA containing 4-sulfamoylbenzoic acid but lacking a peptide sequence ΔC T The almost complete absence of value enrichment confirmed that peptide-mediated barcode hybridization is important for double-strand stabilization. DNA barcodes lacking both pharmacophores and peptide linkers have a measurable ΔC T Since no signal was detected, the possibility of a non-specific amplification product was ruled out (Figure 3E). These control experiment results comprehensively demonstrate that the selectivity of PEMAG-DEL is expressed by an interaction synergy that integrates aptamer-mediated target localization, pharmacological binding affinity, and peptide-barcode hybridization.

[0125] 5. Verification of Aptamer-Dependent Selectivity Mechanism

[0126] To clearly demonstrate the critical role of aptamers in PEMAG-DEL, a selectivity control experiment was performed in which the target protein (CA II) was directly reacted with a positive control (PC) and a negative control (NC) without an aptamer-mediated selectivity step. In this experimental setup, due to the lack of spatial and structural guidance from aptamers, ΔC in the RT-qPCR analysis T The value is ΔC in the presence of aptamer T ΔC compared to = 7 T= decreased to 3 (Figure 3A). This significant reduction in signal difference highlights the essential role of aptamers in inducing target-selective hybridization and the enrichment of high-affinity complexes. Furthermore, in the elemental PAGE analysis, nearly identical band intensities were observed in both PC and NC in the absence of aptamers, implying a loss of specificity and confirming that aptamers are essential for distinguishing between target-binding and non-binding ligands (Figure 3B). These results demonstrate that aptamers function as molecular gatekeepers and spatial organizers, ensuring that only target-proximate small molecules (SMs) are stabilized and amplified. This experiment is a significant result that directly verifies our hypothesis, proving that aptamers are not merely passive components but active factors that activate target-selective DEL screening (Figure 3D). Aptamers [imply] the sensitivity of the analysis (ΔC T By demonstrating that it simultaneously improves ) and specificity (band intensity discrimination), we provide strong grounds for integrating aptamers into the DEL workflow. Additionally, to further verify aptamer selectivity, we selected aptamers specific to EGFR protein as negative aptamers. In this negative control experiment, we hypothesized that using EGFR-specific aptamers for CA II screening would naturally result in the failure to recognize CA II; indeed, crossover experiments with non-specific aptamers (EGFR) against CA II yielded negative results or C T The value changed significantly (Figure 3C). This result demonstrates the specificity and reliability of the system.

[0127] 6. DEL Library Screening and Candidate Ligand Discovery

[0128] To determine the optimal ligand composition for CA II inhibition, a DNA-encoding library (DEL) composed of 125 unique small molecules (SM) was synthesized using split-pool combinatorial chemistry (33-35) (Fig. 6). Each SM is configured with one of five amino acids (leucine, alanine, histidine, glycine, tyrosine) attached via a peptide linker modularized to 4-sulfamoylbenzoic acid. It was hypothesized that the amino acid side chains would form bioorthogonal interactions, such as hydrogen bonding and hydrophobic packing, with the active site of CA II, thereby synergistically enhancing the binding affinity and specificity of sulfonamides under physiological conditions.

[0129] Library screening was performed in a lysis-like environment using PEMAG-DEL. Following aptamer-mediated CA II localization, DEL (125 species) was introduced to enable toehold hybridization only for target-binding SMs. DEL (125 species) has a lower C than PC. T The values ​​indicated that it contained a ligand with higher target affinity for CA II (Figure 3E). A competitive RT-qPCR strategy was applied to preferentially screen high-affinity conjugates. Serial dilution of concentrated DEL (up to 10 6 The primer concentration was gradually reduced (10 μM → 1 μM) along with a factor dilution. This approach resulted in a low primer / template (DEL) concentration (10 6 Dynamic selective pressure is applied so that only the most abundant (i.e., having the highest affinity) SM-aptamer complex is selectively amplified under conditions of 1 / 1 dilution, 1 μM primer). 10 6DNA amplified from the 1 / 2 dilution group was used for Sanger sequencing (Fig. 10), and the obtained barcode sequences were subjected to multiple sequence alignment against DEL's predefined codon-amino acid map using Clustal Omega. This analysis confirmed the dominant SM configurations in which 4-sulfamoylbenzoic acid was bound to Histidine-Histidine-Alanine (Sulfo-His-His-Ala) and Histidine-Histidine-Leucine (Sulfo-His-His-Leu), which exhibited over 90% sequence similarity to the top enrichment barcode (Fig. 11). By integrating aptamer-induced target localization, competitive PCR-based enrichment, and codon-based sequencing, PEMAG-DEL successfully identified high-affinity ligand variants in the combination library.

[0130] 7. RT-qPCR-based validation of selected candidate ligands

[0131] After high-affinity CA II ligands were identified in the 125-type DEL library, the upstream peptide-sulfonamide conjugates Leucine-Histidine-Histidine (Sulfo-His-His-Leu) and Histidine-Histidine-Alanine (Sulfo-His-His-Ala) were validated using RT-qPCR in both solution and cell lysate environments. These candidates were compared to two negative controls: (1) Sulfo-Gly-Ala-Gly, a low-similarity peptide with less than 10% sequence similarity in DEL screening, and (2) a sulfonamide monodermabrasion derivative lacking a peptide chain. Notably, in HeLa cell lysates, the Sulfo-His-His-Leu conjugate C T 19.9, Sulfo-His-His-Ala is C T It demonstrated resistance to molecular congestion and non-specific interference by maintaining 19.6. This performance was demonstrated in C in cell lysate. TThis stands in stark contrast to the Sulfo-Gly-Ala-Gly conjugate, which showed = 24.9, highlighting the importance of peptide-mediated interactions in intracellular ligand efficacy (Fig. 4A). In solution-phase analysis as well, the Sulfo-His-His-Leu and Sulfo-His-His-Ala conjugates were C T Significant values ​​of 18.3 and 20 were shown (NC, C T = 37 (compared to), confirmed strong target binding. In contrast, the Sulfo-Gly-Ala-Gly conjugate and the Sulfonamide monoderived were C, respectively. T 25.9(GAG) and C T It changed to 26(Sulfo), which was consistent with low enrichment results during DEL screening (Figure 4B). These results demonstrate the ability of PEMAG-DEL to select ligands that possess both high affinity and physiological suitability, while also highlighting the critical role of the peptide chain in enhancing sulfonamide-target binding under nature-like conditions.

[0132] 8. Evaluation of biochemical activity and verification of efficacy of selected ligands

[0133] We demonstrated that PEMAG-DEL can function as a proof-of-concept method for directly enriching and identifying small molecule ligands in complex biological environments. Among a library of 125 sulfonamide-peptides screened against Carbonic Anhydrase II (CA II), two conjugates, namely Sulfo-HHA (Histidine-Histidine-Alanine) and Sulfo-HHL (Histidine-Histidine-Leucine), were identified as the most active ligands. These candidates were subsequently evaluated via CA II enzyme inhibition assays, resulting in IC50 values ​​of 0.84 μM (Fig. 5C) and 0.98 μM (Fig. 5B), respectively. 50 Representing the value, IC 50It showed that the binding affinity was significantly improved compared to the unmodified original sulfonamide at 13.8 μM (Fig. 5D). These results indicate a higher C T Although the value suggests that it may be correlated with a stronger inhibitory effect, since there are only two verified hits to date, ΔC T It is premature to conclude a definitive correlation between the inhibitory effect and the inhibitory effect, and systematic screening of a larger library is essential to verify this. The representative image of the 96-well plate (Figure 9) shows a gradual change in the color of the reaction product as the inhibitor concentration increases. A complementary thermal-shift assay showed that the melting temperatures of CA II bound to Sulfo-HHA and Sulfo-HHL were approximately 61 °C and 59 °C, respectively, which is a significant increase compared to 54 °C of the original compound (Figure 10), qualitatively supporting the qPCR-based screening results.

[0134] 9. Verification of universality targeting PD-L1

[0135] To evaluate the universality of PEMAG-DEL, the screening strategy was extended to include the important target PD-L1 (Programmed Death Ligand-1). In this experiment, DNA aptamers with nanomolar affinity were used, with BMS-8-Leu₃ designated as the positive control and Benzoic acid-Leu₃ as the negative control (Figure 12). The analysis was performed under the same conditions as the CA II study. As a result, ΔC between the positive and negative controls in both the buffer and 5% HeLa lysis solutions T A distinct signal difference of = 6.5 was observed. In contrast, in the control group using non-recognition aptamers (CA II) or off-target proteins (BSA), ΔC TThe values ​​were found to be very low, ranging from 1 to 2.8 (Figure 13), confirming aptamer-dependent specificity. In additional negative control experiments, high C was also observed when the core binding component was lacking. T Values ​​appeared, supporting these results.

[0136] Although these findings are limited to RT-qPCR and gel-based data, they demonstrate that the core mechanisms of PEMAG-DEL (spatial gating, accuracy filtering, primer extension) operate effectively beyond a single target. In particular, the distinct ΔC in PD-L1 screening T The differences demonstrate the flexibility of the platform and suggest its applicability to various proteins. These initial results lay the foundation for future research, including complete DEL screening and hit validation for various therapeutically important targets. Furthermore, these findings provide an important basis for refining PEMAG-DEL and designing physiologically relevant ligands under conditions similar to natural environments.

[0137]

[0138] II. Probing Drug-Target Engagement in Living Cells with an Aptamer-Gated DNA-Encoded Library

[0139] Experimental method

[0140] 1. Cell Culture and PD-L1 Aptamer

[0141] MDA-MB-231 human breast cancer cells (TNBC model) were cultured under standard conditions. This cell line was selected due to its high basal expression of PD-L1. DNA aptamers were commercially purchased, and primers with complementary sequences containing the 3′ ends of the aptamers were designed for qPCR and primer extension. Binding buffer and washing conditions were experimentally optimized.

[0142] 2. Aptamer Binding Optimization

[0143] Live MDA-MB-231 cells were seeded into T-25 flasks. After 24 hours, the medium was removed and washed with PBS; following trypsin treatment, the cells were suspended in 100 μL of sterile PBS (pH 7.2) and collected in 1.5 mL Eppendorf tubes. Subsequently, aptamers of various concentrations (15.6 nM–5000 nM) containing Mg 2+ The cells were added to a PBS-based binding buffer containing [specific component] and incubated at 37 °C for 2 hours (based on preliminary time-course results). In parallel experiments, the aptamer concentration was fixed at 500 nM, and incubation times (30 min, 1 hour, 1.5 hours, and 2 hours) were varied. After incubation, cells were washed with PBS to remove unbound aptamers, and cells were isolated after treatment at 95 °C for 10 minutes. The supernatant (containing cell-bound aptamers) was used for qPCR analysis using aptamer-specific primers, and Ct values ​​were plotted to compare signal differences between conditions. Additionally, a portion of the aptamer-cell complex was developed on a 10% urea gel (native PAGE) and stained with EtBr to verify the presence of aptamers (changes in mobility indicated bound aptamers). Through this analysis, the optimal aptamer concentration and incubation time to induce maximum binding were established.

[0144] 3. DEL Screening Protocol in Living Cells

[0145] DEL screening was performed according to the Primer-Extension Mediated Aptamer-Gated DEL (PEMAG-DEL) protocol. MDA-MB-231 cells (approx. 1.5 × 10⁶ per sample) 6The cells were inoculated into a T-25 flask, and after trypsin treatment, suspended in 100 μL of sterile PBS (pH 7.2) and collected in a 1.5 mL Eppendorf tube. Subsequently, 500 nM aptamer was added and incubated at 37 °C for 30 minutes to allow the aptamer to bind to PD-L1. Then, the positive control ligand BMS-8 (a well-known PD-L1 inhibitor) and the negative control ligand benzoate (Benzo, a non-binding analog) were added at a concentration of 10 μM each, and the cells were treated at 37 °C for 1–1.5 hours. Afterward, Exonuclease I (Exo I, 20 U) was directly added to the cell suspension and reacted at 37 °C for 1.5 hours to degrade unbound single-stranded DNA (unbound DEL tags or primers). Exo I was then inactivated by treatment at 80 °C for 20 minutes. A primer extension mixture (2 U nPfu DNA polymerase, extension primer) was added, and the cells were incubated at 37 °C for 2 hours to replicate the barcode from the aptamer primer to the DEL tag. Subsequently, a termination reaction to ligate the common adapter was performed overnight using T4 DNA ligase (800 U). Cells were treated at 95 °C to detach the DEL tag, and the supernatant was collected after centrifugation; qPCR was then performed targeting the adapter-linked DNA. The difference in qPCR signals between the positive control (BMS-8) and negative control (Benzo) conditions indicates PD-L1 target binding. All experiments were performed in at least three replicates.

[0146] 4. Gel Electrophoresis and Imaging

[0147] A portion of the primer extension products was developed on a 10% urea gel (urea-PAGE) and stained with EtBr to visualize the PCR products. The estimated product size was approximately 140 bp (primer + adapter). For fluorescence imaging, FAM-labeled PC and NC were applied to cells, and images were taken using a fluorescence microscope after washing. Aptamer localization was analyzed by overlaying the brightfield and green (FAM) channels, and aptamer / DEL binding was visually verified.

[0148]

[0149] result

[0150] 1. Aptamer binding optimization

[0151] qPCR analysis of aptamer binding to living cells showed a distinct increase in signal with increasing aptamer concentration (Figure 17). The signal increased rapidly in the 0–250 nM range and saturated at 250–500 nM. The highest amplification signals were observed at 250 nM and 500 nM, indicating that these concentrations are close to the aptamer dissociation constant (Kd). At concentrations below 30 nM, almost no signal was observed, and at high concentrations above 1000 nM, the increase was limited due to non-specific false positives (background signals). Therefore, it was confirmed that the optimal aptamer concentration range is approximately 250–500 nM.

[0152] Similarly, varying culture times at a fixed aptamer concentration confirmed that binding improved with increasing culture time (Figure 18). The signal nearly doubled from 30 minutes to 1.5 hours and reached a maximum at 2 hours. Based on these results, an aptamer concentration of 500 nM and a culture time of 30 minutes were selected as the standard binding conditions for subsequent experiments. Under these conditions, aptamer binding to MDA-MB-231 cells was consistently detected via qPCR and PAGE. In urea-PAGE analysis (data not shown), aptamer bands were observed in cell pellet samples cultured for 2 hours, whereas no bands appeared in the aptamer-free control group. Therefore, it was confirmed that the aptamer specifically binds to PD-L1-expressing cells under optimized conditions (500 nM, 30 minutes).

[0153] 2. Live Cell DEL Screening and qPCR Analysis

[0154] In the PEMAG-DEL protocol, the DEL binding effect was measured by treating MDA-MB-231 cells with aptamers followed by PD-L1 ligands. Under the positive control (PC) condition, cells were exposed to the PD-L1 inhibitor BMS-8, while under the negative control (NC) condition, benzoate was treated. Following Exo I treatment and primer extension, qPCR results showed that the difference in Ct values ​​between the PC and NC conditions was not significant, at the 1-2 cycle level; however, in all three replicate experiments, the PC condition exhibited a higher signal than the NC condition. This suggests that BMS-8 acted competitively on PD-L1 binding, resulting in reduced DEL recovery under the NC condition. Although fold-enrichment was limited, this demonstrates that the method possesses proof-of-concept validity in protein-based systems. In the future, expanding DEL diversity and optimizing amplification cycles could amplify the signal difference.

[0155] 3. Gel electrophoretic analysis of primer extension products

[0156] PAGE analysis of the final PCR products revealed a band of the expected size (~140 bp) in the PC (BMS-8) sample (Figure 19). In the NC (benzoate) sample, only a faint band was observed at the same location. Overall, the bands were broad and low in intensity, resulting in limited resolution; however, the appearance of the expected band only in the PC sample indicates that the primer extension and linkage reactions were successfully performed. Therefore, the gel electrophoresis results are consistent with the qPCR data and support the selective replication of the DEL tag under PD-L1 target conditions.

[0157] 4. Fluorescence Microscopy Analysis

[0158] Fluorescence imaging results showed that a strong green fluorescent signal was localized on the cell surface under PC conditions (Fig. 20A). In contrast, almost no fluorescent signal was observed in NC (benzoate) cells (Fig. 20B). This visually demonstrates the qPCR trend and suggests that while the aptamer-DEL complex remains bound to the cell under conditions where PD-L1 is not blocked, binding is blocked due to competitive binding upon benzoate treatment.

Claims

1. A step of reacting a target protein, a candidate ligand, and an aptamer that binds to the target protein in a biological sample; and The method comprises the step of selecting a candidate ligand that forms a target protein-aptamer-candidate ligand complex through the above reaction; The above candidate ligand includes a DNA barcode, and A method for selecting a target protein-specific ligand, wherein the aptamer comprises a DNA sequence capable of binding complementarily to the DNA barcode of a candidate ligand.

2. A method for selecting a target protein-specific ligand according to claim 1, wherein the biological sample comprises a cell or a cell lysate.

3. A method for selecting a target protein-specific ligand according to claim 1, wherein the candidate ligand is in a form in which a DNA barcode is covalently bonded to an amino acid, peptide, protein, or small molecule.

4. A method for screening target protein-specific ligands according to claim 1, further comprising the step of removing candidate ligands that do not form a target protein-aptamer-candidate ligand complex.

5. A method for selecting a target protein-specific ligand according to claim 4, wherein the removing step is performed by a nuclease.

6. A method for selecting a target protein-specific ligand according to claim 1, wherein the step of selecting the candidate ligand is performed by forming an amplification product by performing primer extension on the complex, and then performing quantitative analysis of the amplification product.

7. A method for selecting a target protein-specific ligand according to claim 6, wherein the primer extension is performed using nPfu DNA polymerase. 8.(a) Aptamer that binds to a target protein, (b) candidate ligands containing a DNA barcode, and (c) comprising a nuclease, a primer, and a polymerase, and A kit for selecting target protein-specific ligands, wherein the aptamer comprises a DNA sequence capable of binding complementarily to the DNA barcode of the candidate ligand.

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