Cell membrane in-situ drug membrane protein target screening method based on variable configuration DNA

Abstract

The invention discloses a cell membrane in-situ drug membrane protein target screening method based on variable configuration DNA, and belongs to the technical field of biological medicine. Two pairs of functionalized nucleic acid structures are constructed, the first pair is S1S2 semi-complementary DNA double strands, the second pair is S3S4 DNA-RNA hybrid double strands, and the S1S2 structure is covalently coupled to a non-natural amino acid site-directed modified membrane protein through a click chemical reaction in a living cell to realize tagging of the membrane protein; when the candidate drug coupled in S3 interacts with the membrane protein, triggering an allosteric response system to release the tagged chain in S1; the released DNA tag is subjected to PCR amplification and sequencing, and accurate identification and analysis of a membrane protein target are realized according to a tag sequence. The invention provides a high-specificity and non-in-vitro drug membrane protein target screening technology. According to the technology, active compound discovery or accurate screening of drug membrane protein targets in a living cell in-situ environment can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for screening cell membrane protein targets for in situ drug delivery based on allosteric DNA. Background Technology

[0002] Drug target screening and validation are crucial steps in the new drug development process. With the rapid advancements in organic synthesis methodologies and compound separation technologies, a large number of small-molecule bioactive compounds derived from natural products have emerged, possessing novel skeletons and structures. In new drug development, identifying the targets and molecular mechanisms of bioactive compounds is not only a core step in the research and development process but also an important basis for assessing drug safety (such as predicting toxic side effects) and ensuring rational clinical use.

[0003] Membrane proteins, as a crucial class of drug targets, possess vital biological functions and participate in numerous important processes within cells and organisms. Many diseases are closely related to abnormal membrane protein function. Data shows that over 60% of approved drugs target cell surface membrane proteins. However, target screening research for membrane proteins faces significant challenges. First, bottlenecks remain in biotechnologies such as membrane protein expression and purification. Second, the biological activity of membrane proteins heavily depends on the three-dimensional conformation of the cell membrane, and their function may be lost or altered in the extracellular environment. Currently, commonly used protein target screening methods primarily rely on proteomics techniques based on mass spectrometry, utilizing active molecules to target specific proteins in cell lysates or expressed and purified proteins, followed by mass spectrometry analysis to identify the target proteins. However, it is noteworthy that these methods mostly screen intracellular proteins, and due to the cellular environment-dependent activity of membrane proteins and the challenges of expression and purification, they rarely address the screening of membrane protein targets. Therefore, developing a new method adapted to the natural cellular environment and capable of effectively screening membrane protein targets has become an urgent need in the field of membrane protein research.

[0004] DNA possesses an allosteric switch effect for in-situ molecular recognition and, as an information carrier, has an enormous storage capacity. If we consider four nucleotides as encoding, a short DNA chain of only 10 nucleotides can generate over a million (4...) codes. 10 =1048576) permutations and combinations. Therefore, using DNA as a coding tag to label specific molecules allows for precise identification in complex systems through DNA sequence amplification and high-throughput sequencing, with extremely high sensitivity. Based on this, DNA-encoded chemical library (DEL) technology has emerged and has become one of the cutting-edge technologies in the field of new drug discovery.

[0005] DEL technology can quickly and efficiently construct and screen libraries of hundreds of millions of molecules, demonstrating significant time and cost advantages in the discovery of small molecule lead compounds. However, existing DEL technologies mainly target specific proteins and are typically applied to purified in vitro expressed proteins, with intracellular proteins being the primary focus of research. Therefore, DEL technology has not effectively addressed the challenges of screening membrane protein targets, especially the identification and screening of membrane proteins in the in situ cellular environment. Summary of the Invention

[0006] Purpose of the invention: Given the research value of membrane proteins as important drug targets and the inadequacy of existing target screening methods for membrane proteins, this invention provides a cell membrane in situ drug screening method based on allosteric DNA, taking full advantage of the specificity and information storage of DNA tags. This method utilizes nucleic acid chemical biology strategies to achieve the labeling and detection of membrane proteins in a living cell environment, and constructs a drug-triggered membrane protein target screening platform.

[0007] The screening method of this invention can solve the problems of difficult expression and purification of membrane proteins and easy loss of in vitro activity in the prior art, effectively address the problem of membrane protein identification and screening, and provide a precise, flexible and efficient solution for drug development.

[0008] Technical solution: The objective of this invention is achieved through the following technical solution: This invention provides a method for screening cell membrane protein targets for in situ drug delivery based on allosteric DNA, comprising the following steps: (1) Two pairs of functionalized nucleic acid structures for membrane protein tag release are constructed. The first pair is a S1S2 semi-complementary DNA double strand, in which the S1 strand is the membrane protein tag strand and the S2 strand is the functional strand coupled with the membrane protein ligand. The two form a dynamic and reversible double strand structure through semi-complementary pairing. The second pair is a S3S4 DNA-RNA hybrid double strand, in which the S3 strand is coupled with the candidate drug and the S4 strand is the RNA strand complementary to the S3 strand. The present invention constructs two pairs of functionalized nucleic acid structures to achieve the specific release of nucleic acid tags of membrane proteins using a logic gate strategy. (2) In living cells, non-natural amino acids are introduced into specific sites of target membrane proteins through genetic code expansion technology (GCE), and bioorthogonal reactions occur to form modifiable sites; The non-natural amino acids include fluorosulfonic acid-L-tyrosine (FSY), fluorosulfonyloxybenzoyl lysine (FSK), acetyl lysine (AcK), or azide lysine (AzK), etc.

[0009] (3) Using click chemistry, the S2 strand in the S1S2 semi-complementary DNA double-stranded structure is covalently coupled to the target membrane protein to achieve the labeling of the membrane protein; (4) Introduce the S3S4 DNA-RNA hybrid double-stranded structure into the living cell environment. When the candidate drug coupled with the S3 chain binds to the target membrane protein, it induces ribonuclease to mediate the cleavage of the S4 chain, so that the S3S4 structure becomes an S3 single strand. (5) The S3 single strand then undergoes a DNA strand substitution reaction with the S1S2 semi-complementary DNA double strand on the membrane protein to form the S2S3 fully complementary DNA double strand, releasing the S1 tag strand. (6) The released S1 tag chain is amplified by PCR and the tag sequence is obtained by Sanger / high-throughput sequencing. The target membrane protein is identified and analyzed based on the sequence information.

[0010] This invention constructs fluorescently labeled S1S2 semi-complementary DNA double strands and drug-conjugated S3S4 DNA-RNA hybrid double strands, and performs target screening in situ in living cells via DNA strand displacement reaction.

[0011] The S1 chain contains coding information that uniquely matches the structure of its corresponding membrane protein. This coding information can be read and decoded through PCR amplification and DNA sequencing.

[0012] Preferably, in step (1), the S1 chain includes a partially complementary region and a coding region; the coding region sequence is a specific DNA sequence encoding a membrane protein, which varies depending on the membrane protein. The S1 strand DNA sequence is 80–110 bases long; and / or, the S2 strand DNA is 40–60 bases long; and / or, the S3 strand DNA is 50–70 bases long; and / or, the S4 strand RNA is 30–50 bases long; wherein the complementary base pairing length of the S1 and S2 strands is less than the complementary base pairing length of the S2 and S3 strands. The 5' end of the S2 chain is modified with a functional group that couples with the membrane protein ligand, including any one of amino, dibenzocyclooctylene, thiol, and maleimide. Depending on the membrane protein ligand (usually including antibodies, nanobodies, peptides, aptamers, etc.), a functional group that reacts with a specific functional group is modified at the 5' end of the S2 chain, and the membrane protein ligand is coupled to the modified 5' end of the S2 chain through a click chemistry reaction.

[0013] The 3' end of the S3 chain is modified with a functional group that is coupled to the candidate drug, including any one of amino, dibenzocyclooctylene, mercapto, and maleimide.

[0014] Preferably, the S3 chain is connected to a coupling group, and one of azide, maleimide, NHS ester, and thiol is modified at the inactive site of the candidate drug. The coupling group in the S3 chain is then combined with the candidate drug molecule through a click chemistry reaction.

[0015] In step (5), the DNA strand substitution reaction (i.e., the allosteric response system) is achieved by spatial proximity-induced ribonuclease cleavage, and the reaction process is dependent on membrane protein-ligand interaction.

[0016] The present invention also provides a specific preferred embodiment, wherein the screening method includes the following steps: (1) A fluorescent group is modified at the 3' end of the S1 chain to form a "fluorescent group-S1 chain"; A membrane protein ligand is coupled to the 5' end of the S2 chain to form a "membrane protein ligand-S2 chain". A dynamically reversible S1S2 semi-complementary DNA double strand is formed through the complementary pairing segment between the "fluorescent group-S1 chain" and the "membrane protein ligand-S2 chain". (2) The candidate drug is coupled to the 3' end of the S3 chain to form a "drug-S3 chain"; the "drug-S3 chain" is complementary to the RNA chain S4 to form an S3S4 DNA-RNA hybrid double strand; (3) In living cells, non-natural amino acids are introduced into specific sites of target membrane proteins using GCE technology to stably express non-natural amino acid-modified membrane proteins; (3) Cells that stably express non-natural amino acid-modified membrane proteins are co-incubated with S1S2 semi-complementary DNA double strands, so that they are anchored to the membrane surface through covalent binding between membrane protein ligands and special reactive functional groups on non-natural amino acids. (4) Introduce the S3S4 DNA-RNA hybrid double-stranded structure into the living cell environment. When the candidate drug coupled with the S3 chain binds to the target membrane protein, in order to avoid false positive results, add ribonuclease to degrade the RNA chain S4, trigger the DNA chain replacement reaction, so that the "membrane protein ligand-S2 chain" and the "drug-S3 chain" are completely complementary and pair, thereby releasing the "fluorescent group-S1 chain". (5) The "fluorescent group-S1 chain" was analyzed by PCR amplification and Sanger / high-throughput sequencing, and the membrane protein target information was reverse mapped.

[0017] Furthermore, the nucleotide sequence of the "fluorescent group-S1 chain" and the complementary pairing region of the "membrane protein ligand-S2 chain" contain a complementary sequence of 20-35 bp; the complementary pairing region of the "membrane protein ligand-S2 chain" and the "drug-S3 chain" contains a complementary sequence of 40-60 bp.

[0018] Furthermore, the S1 chain is modified with the fluorescent group Cy3, and its sequence is shown in SEQ ID NO.1; the S2 chain is a DNA chain with a DBCO group modified at the 5' end, and its sequence is shown in SEQ ID NO.3; the S3 chain is modified with the DBCO group, and its sequence is shown in SEQ ID NO.5; the sequence of the S4 chain is shown in SEQ ID NO.6.

[0019] Preferably, the two pairs of functionalized nucleic acid structures are constructed using a thermal annealing procedure, wherein the thermal annealing procedure is as follows: ① Reaction at 95℃ for 5 minutes; ② Reaction at 4℃ for 30 minutes.

[0020] Furthermore, the present invention also provides a method for constructing S1S2 semi-complementary DNA double strands, comprising the following steps: (1) Dissolve the S1 chain dry powder modified with the fluorescent group Cy3 (the sequence is shown in SEQ ID NO.1) in PBS buffer to obtain Cy3-S1 chain solution; (2) Dissolve the SLC7A5 membrane protein ligand-peptide (sequence shown in SEQ ID NO.4) in PBS to prepare a peptide solution; dissolve the DBCO-modified DNA S2 chain dry powder (sequence shown in SEQ ID NO.3) in PBS to obtain a DBCO-S2 chain solution; The polypeptide and DBCO-S2 chain were added to the polypeptide solution and DBCO-S2 chain solution at a molar ratio of 10:1 and reacted by click chemical reaction; the unreacted S2 chain was removed by purification to obtain the purified "membrane protein polypeptide ligand-S2 chain"; (3) The obtained “membrane protein polypeptide ligand-S2 chain” and Cy3-S1 chain are mixed in an equimolar ratio and formed S1S2 semi-complementary DNA double strands by thermal annealing. The heat annealing process is as follows: ① Reaction at 95℃ for 5 minutes; ② Reaction at 4℃ for 30 minutes.

[0021] Furthermore, the present invention also provides a method for constructing an S3S4 DNA-RNA hybrid double strand, comprising the following steps: (1) Dissolve the S3 chain modified with DBCO group (sequence shown in SEQ ID NO.5) in PBS buffer to obtain DBCO-S3 chain solution; (2) The azide-modified SLC7A5 inhibitor Diiodo-Tyr was dissolved in DMSO to obtain a Diiodo-Tyr solution; (3) Mix DBCO-S3 chain and Diiodo-Tyr in equimolar ratio, add DBCO-S3 chain solution and Diiodo-Tyr solution, and after click chemical reaction, obtain S3 chain coupled with Diiodo-Tyr, namely "TYR-S3 chain"; (3) Mix “TYR-S3 strand” and S4 strand RNA (sequence shown in SEQ ID NO.6) in an equimolar ratio and form S3S4 DNA-RNA hybrid double strands by thermal annealing. The heat annealing procedure is as follows: ① 95℃, react for 5 min; ② 4℃, react for 30 min.

[0022] Taking the membrane protein SLC7A5 as an example, this invention also provides a method for introducing non-natural amino acids into specific sites of the target membrane protein in living cells using genetic code expansion technology (GCE) in step (2), comprising the following steps: 1. Plasmid construction: Construct expression plasmid pcDNA-SLC7A5-220TAG-C-eGFP; 2. Cell preparation: Seed and culture live cells until the cell confluence reaches 70%-80%; 3. Preparation of transfection complex: The transfection reagent was added to serum-free culture medium to obtain liposome dilution. The pcDNA3.1(+)_SLC7A5-220TAG_-C-eGFP plasmid and pNEU-FSYRS plasmid were dissolved in the culture medium to obtain plasmid dilution. The plasmid dilution and liposome dilution were mixed to form plasmid DNA-Lipofectamine transfection complex. 4. Live cell transfection expression: After culturing the transfection complex, remove the transfection mixture; add medium containing fluoxetine-L-tyrosine to the mixture for further culture to express the SLC7A5 membrane protein modified with FSY.

[0023] Preferably, in step (3), the target membrane protein is one or more human cell surface membrane proteins that have been modified with non-natural amino acids, such as SLC7A5, SLC1A5, SLC38A1, SLC38A2, ABCC4, etc.

[0024] Preferably, the polymerase used in the PCR amplification reaction includes one of 2×Taq plus PCR Master Mix, Platinum™ SuperFi II PCR Master Mix, and 2×Phanta Max Master Mix, and the DNA sequence length of the upstream and downstream primers is 19 to 22 bases.

[0025] Furthermore, the PCR amplification reaction program is as follows: ① pre-denaturation, 95℃ for 5 min; ② denaturation, 95℃ for 15 sec; ③ annealing, 52℃ for 15 sec; ④ extension, 72℃ for 30 sec; repeat steps ② to ④ for a total of 45 to 50 cycles; finally, extend at 72℃ for 5 to 10 min.

[0026] The screening method of this invention can screen drug targets in situ in living cells, and has high specificity and high sensitivity. It is suitable for functional verification of membrane proteins, screening of drug targets, and development research of new targets.

[0027] The in-situ drug target screening method for cell membranes based on allosteric DNA structure described above in this invention can be used for new drug discovery.

[0028] The in-situ drug target screening method for cell membranes based on the modified DNA structure described above can also be used for the discovery of membrane protein targets. Beneficial effects

[0029] 1. This invention provides a method for in-situ drug screening of membrane protein targets based on allosteric DNA, emphasizing direct labeling and detection of membrane proteins in living cells. Two pairs of functionalized nucleic acid structures are designed to undergo DNA allosteric transformation via strand displacement reactions, releasing membrane protein tag chains. These tag chains are amplified by PCR and sequenced to resolve their sequence information, enabling precise target identification. This technology allows for accurate screening of drug membrane protein targets in a living cell in-situ environment.

[0030] 2. This invention uses a touch-controlled DNA allosteric strategy to screen drug membrane protein targets in situ in living cells. By utilizing DNA allosteric reactions triggered by ribonuclease, the interference of false positive results caused by random collisions is greatly reduced, the specificity of membrane protein target screening is improved, and the technical bottleneck of DEL technology in the field of drug membrane proteins is solved. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the DNA allosteric strategy constructed in this invention; Figure 2 PAGE characterization of self-assembly of allosteric DNA molecules and enzyme-stimulated responsive DNA allostericity; Figure 3 Subcellular localization using a DNA allosteric strategy; the figure illustrates the cellular localization process of the SLC7A5 membrane protein modified with fluorosulfonic acid-L-tyrosine after binding to labeled DNA allosteric molecules using this strategy. Figure 4 This is a schematic diagram illustrating the application principle of the DNA allosteric strategy of the present invention in in-situ screening of membrane protein targets in living cells; the diagram shows the identification and screening of membrane protein targets through a DNA strand displacement reaction. Figure 5 Gel electrophoretic characterization of in situ membrane protein target screening based on allosteric DNA; Figure 6 The image shows the Sanger sequencing results for in situ screening of membrane protein targets based on allosteric DNA; the figure shows the sequencing results of DNA after PCR amplification and its corresponding membrane protein target information. Detailed Implementation

[0032] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0033] Unless otherwise specified, the reagents and instruments used in the examples are conventionally selected in the art. Experimental methods not specifying particular conditions in the examples were performed under standard conditions.

[0034] A schematic diagram of the DNA allosteric strategy constructed in this invention is shown below. Figure 1 This diagram illustrates the basic principle of screening membrane protein targets through allosteric DNA structure.

[0035] In this design, the 3' end of the S1 strand can be coupled with a fluorescent group, the 5' end of the S2 strand is coupled with a membrane protein ligand, the 3' end of the S3 strand is coupled with a candidate drug, and the S4 strand is an RNA strand. Two pairs of functionalized nucleic acid structures were designed: an S1S2 semi-complementary DNA double strand and an S3S4 DNA-RNA hybrid double strand. When RNase was added to the system, the S4 strand degraded, and the S3 single strand subsequently underwent a DNA strand displacement reaction with the S1S2 semi-complementary DNA double strand to form an S2S3 fully complementary DNA double strand, releasing the S1 tag strand.

[0036] Comparative Example 1: Preparation of S1S2 semi-complementary DNA double strands (Method 1) (1) The Cy3-modified S1 chain dry powder (Cy3-S1 chain) and the thiol-modified S2 chain dry powder (HS-S2 chain) were purchased from Sangon Biotech (Shanghai) Co., Ltd. The above dry powders were dissolved in PBS buffer according to the instructions to obtain Cy3-S1 chain and HS-S2 chain solutions with a concentration of 100 uM, the sequences of which are shown in SEQ ID NO.1-2.

[0037] The Cy3-S1 chain shown in SEQ ID NO.1 has a Cy3 fluorescent group modified at its 3' end: 5'-TCTTCTAACCTCTCACTCGTCTACACAGCAAATCCGATATCAGAACCGTCAAACCCCTTAAGTCCTCCGCAAACCTTATC-Cy3-3'; The HS-S2 chain shown in SEQ ID NO.2 has a thiol group modified at its 5' end: 5'-HS-CATTAGAACGGATAAGGTTTGCCGGAGGACTTAAGGGGTAA-3'.

[0038] (2) SLC1A5 membrane protein ligand-antibody (20350-1-AP, 1 μg / μL, Wuhan Sanying Biotechnology Co., Ltd.), take 50 μL and dissolve it in PBS (pH 7.4) to prepare 100 μL antibody solution.

[0039] The crosslinking agent 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC, Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in ddH2O to prepare a 200μM solution for later use.

[0040] The above antibody solution was reacted with 100 μL of SMCC solution (200 μM) in a metal bath at 37°C and 800 rpm for 2 h. The mixture was then purified using ultrafiltration buffer and a 10 kDa ultrafiltration tube to remove excess SMCC, yielding the purified antibody-SMCC intermediate. The ultrafiltration buffer was PBS (pH 7.4) containing a final concentration of 1 mM EDTA.

[0041] (3) The purified antibody-SMCC intermediate was mixed with the thiol-modified S2 chain (HS-S2 chain, 100 μM) in an equimolar ratio and reacted in a metal bath at 28 °C and 800 rpm for 2 h. Then, the unreacted S2 chain was removed by purification using a 50 kDa ultrafiltration tube. The ultrafiltration buffer was PBS (pH 7.4) to obtain the purified “membrane protein antibody ligand-S2 chain”.

[0042] (4) The obtained “membrane protein antibody ligand-S2 chain” and Cy3-S1 chain are mixed in an equal molar ratio and formed into S1S2 semi-complementary DNA double strands by thermal annealing.

[0043] The heat annealing process is as follows: ① Reaction at 95℃ for 5 minutes; ② Reaction at 4℃ for 30 minutes.

[0044] Example 1: Preparation of S1S2 semi-complementary DNA double strands (Method 2) (1) The dry powder of S1 chain modified with fluorescent group Cy3 (Cy3-S1 chain, the sequence of which is shown in SEQ ID NO.1) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0045] DBCO-modified S2 chain dry powder (DBCO-S2 chain, the sequence of which is shown in SEQ ID NO.3) was purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0046] The DBCO-S2 chain shown in SEQ ID NO.3 has a DBCO group modified at its 5' end: 5'-DBCO-CATTAGAACGGATAAGGTTTGCCGGAGGACTTAAGGGGTAA-3'.

[0047] The above dry powder was dissolved in PBS buffer according to the instructions to obtain a Cy3-S1 chain solution and a DBCO-S2 chain mother liquor with a concentration of 100 uM.

[0048] (2) SLC7A5 membrane protein ligand-peptide (hereinafter referred to as "peptide", the sequence of which is shown in SEQ ID NO.4) was purchased from Shanghai Chutai Biotechnology Co., Ltd. 1 mg of SLC7A5 membrane protein ligand-peptide was weighed and dissolved in PBS (pH 7.4) to prepare a 500 μM solution for later use.

[0049] The polypeptide shown in SEQ ID NO.4 has an azide group modified at its C-terminus: His-Tyr-Pro-Ser-Lys(N3).

[0050] Dilute the DBCO-S2 chain mother liquor with PBS (pH 7.4) to a 50 μM solution for later use.

[0051] The peptide and DBCO-S2 chain were added at a molar ratio of 10:1, with 50 μL of peptide solution (500 μM) and 50 μL of DBCO-S2 chain (50 μM) added. The reaction was carried out via click chemistry in a metal bath at 28 °C and 800 rpm for 2 h with shaking. Subsequently, purification was performed using a 50 kDa ultrafiltration tube to remove unreacted S2 chain; the ultrafiltration buffer was PBS (pH 7.4), yielding the purified "membrane protein peptide ligand-S2 chain".

[0052] (3) The obtained “membrane protein polypeptide ligand-S2 chain” is mixed with Cy3-S1 chain in an equimolar ratio and then subjected to thermal annealing to form S1S2 semi-complementary DNA double strand (i.e. Cy3-S1S2-polypeptide).

[0053] The heat annealing process is as follows: ① React at 95℃ for 5 minutes; ② React at 4℃ for 30 minutes.

[0054] Compared to Comparative Example 1, which uses antibody to conjugate S2 to prepare S1S2 semi-complementary DNA double strands, Example 1 of this invention uses conventional conjugated polypeptides. The experimental system constructed by polypeptides is simpler to operate and more cost-effective, without the need for special linkers such as customized antibodies or heterobifunctional crosslinking agents (such as SMCC).

[0055] Example 2 Preparation of S3S4 DNA-RNA hybrid double strands (1) The DBCO-modified S3 chain (DBCO-S3 chain, purchased from Sangon Biotech (Shanghai) Co., Ltd., its sequence is shown in SEQ ID NO.5) was dissolved in PBS (pH 7.4) buffer to prepare a 50 μM solution for later use.

[0056] The S3 chain shown in SEQ ID NO.5 has a DBCO group modified at its 3' end: 5'-TTACCCCTTAAGTCCTCCGCAAACCTTATCGTCCACCTCATCCATATCTAGTATCTCGTT-DBCO-3'.

[0057] (2) The azide-modified SLC7A5 inhibitor Diiodo-Tyr was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and dissolved in DMSO to a final concentration of 500 μM for later use.

[0058] DBCO-S3 chain (50 μL, 50 μM) and Diiodo-Tyr (5 μL, 500 μM) were mixed in an equimolar ratio and reacted in a metal bath at 28 °C and 800 rpm for 2 h with shaking. After a click chemistry reaction, an S3 chain coupled with Diiodo-Tyr, namely "TYR-S3 chain", was obtained.

[0059] (3) Mix “TYR-S3 strand” and S4 strand RNA (purchased from Sangon Biotech (Shanghai) Co., Ltd., whose sequence is shown in SEQ ID NO.6) in an equimolar ratio and form S3S4 DNA-RNA hybrid double strand (i.e. TYR-S3S4) by thermal annealing.

[0060] The heat annealing procedure is as follows: ① 95℃, react for 5 min; ② 4℃, react for 30 min.

[0061] The S4 chain shown in SEQ ID NO.6: 5'-UGAGGUGGACGAUAAGGUUUGCGGAGGACUUAAGGGGUAA-3'.

[0062] Example 3: Self-assembly and enzyme responsiveness of two pairs of functionalized nucleic acid structures (1) Cy3-modified S1 chain (Cy3-S1 chain, hereinafter referred to as "S1 chain"), DBCO-modified S3 chain (DBCO-S3 chain, hereinafter referred to as "S3 chain"), and RNA chain S4 chain were all purchased from Sangon Biotech (Shanghai) Co., Ltd., and DBCO-modified DNA chain S2 (DBCO-S2 chain, hereinafter referred to as "S2 chain") was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. The above-mentioned S1, S2, S3, and S4 chains (dry powder) were dissolved in PBS (pH 7.4) to prepare 20 μM solutions for later use.

[0063] (2) Take 5 μL of each of the above S1 and S2 strands at a concentration of 20 μM and mix them in an equimolar ratio. Then, use a thermal annealing process to form S1S2 semi-complementary DNA double strands.

[0064] Take 5 μL of each of the above S2 and S3 strands at a concentration of 20 μM and mix them in an equimolar ratio. Then, form S2S3 fully complementary DNA double strands by thermal annealing.

[0065] Take 5 μL of each of the above S3 and S4 strands at a concentration of 20 μM and mix them in an equimolar ratio. Then, form S3S4 DNA-RNA hybrid double strands by thermal annealing.

[0066] 5 μL of each of the above S1, S2, S3 and S4 chains at a concentration of 20 μM were mixed in an equimolar ratio and then subjected to a thermal annealing process to form S1S2S3S4.

[0067] Take 5 μL of each of the above S1, S2, S3 and S4 chains with a concentration of 20 μM and mix them in an equimolar ratio. Then, use a thermal annealing program to form S1S2S3S4. Add 4 μL of Reaction Buffer (10X) and 4 μL of RNase H (purchased from APE×BIO) to prepare the S1S2S3S4+RNase H solution.

[0068] The above heat annealing procedures are all: ① 95℃, reaction for 5 min; ② 4℃, reaction for 30 min.

[0069] (3) S1, the synthesized S1S2, S2S3, S3S4, S1S2S3S4 and S1S2S3S4+RNase H were subjected to polyacrylamide gel electrophoresis (PAGE) to characterize their self-assembly process and RNase responsiveness.

[0070] Electrophoresis experiments were performed using 15% electrophoresis gels prepared with TBE solution, ddH2O, APS solution, and TEMED (tetramethylethylenediamine), and run at 120V for 1 h. Staining with SYBR Green I nucleic acid dye was used, and images were captured using a fluorescence imager. PAGE characterization of conformational DNA molecule self-assembly and enzyme-stimulated DNA conformational changes is shown below. Figure 2 This figure reflects the self-assembly of DNA molecules and the RNA chain cleavage reaction induced by RNase H under different reaction conditions.

[0071] like Figure 2 As shown, the experiment demonstrates that the desired nucleic acid molecular structure can be successfully formed, and in the presence of ribonuclease H, the RNA chain is cleaved, the DNA undergoes an allosteric response, generating S2S3 and releasing S1.

[0072] Example 4: Subcellular localization using DNA allosteric strategies The structural file of the membrane protein SLC7A5 was downloaded from the AlphaFold protein structure database. A machine learning model was used to screen for potential FSY integration sites in SLC7A5, simulating that the 220th amino acid in the SLC7A5 gene coding sequence (CDS) was the optimal insertion site for fluorosulfonic acid-L-tyrosine (FSY). The gene encoded at position 220 was mutated to the TAG codon. The expression plasmid pcDNA-SLC7A5-220TAG-C-eGFP was constructed by inserting the HindIII / NotI multiple cloning site into the pcDNA3.1(+) vector. The vector contained an eGFP signal sequence at its C-terminus. The expression plasmid pcDNA-SLC7A5-220TAG-C-eGFP was synthesized by Nanjing GenScript Biotech Co., Ltd. All plasmids used in the experiments were purchased from Nanjing GenScript Biotech.

[0073] An expression system for the SLC7A5 membrane protein expressing the non-natural amino acid fluorosulfonic acid-L-tyrosine (pcDNA-SLC7A5-220TAG-C-eGFP plasmid, pNEU-FSYRS plasmid, fluorosulfonic acid-L-tyrosine) was transfected into human embryonic kidney cells HEK293T (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) for expression to obtain the SLC7A5 membrane protein containing FSY modification. Detailed steps are described in Example 5.

[0074] 48 h after transfection, the cells were washed twice with pre-cooled PBS (pH 7.4), and Cy3-S1S2-peptide (prepared in Example 2, 2.5 μM, 500 μL, PBS as solvent) was added and incubated at room temperature for 30 min. The cells were then washed once more with PBS.

[0075] Untransfected 293T cells were simultaneously incubated with Cy3-S1S2-peptide (prepared in Example 2, 2.5 μM, 500 μL, PBS as solvent) at room temperature for 30 min, and then washed once with PBS.

[0076] Images were captured using a laser confocal microscope, and the results are as follows: Figure 3 As shown in the figure, the cell localization process of the SLC7A5 membrane protein modified with fluorosulfonic acid-L-tyrosine after binding to the labeled DNA allosteric molecule is illustrated by this strategy.

[0077] In the transfected cells, green fluorescence and significant Cy3 fluorescence (red) were successfully detected, verifying the successful modification of fluorosulfonic acid-L-tyrosine at site 220 of the membrane protein SLC7A5, and its covalent binding to the Cy3-S1S2-peptide via a fluorine-sulfur exchange reaction.

[0078] Example 5: Screening and gel characterization of in situ membrane protein targets based on conformational DNA This embodiment applies a conformational DNA strategy to in situ screening of membrane protein targets in the cell membrane. A schematic diagram illustrating the application principle is shown below. Figure 4 As shown, the identification and screening of membrane protein targets are achieved through DNA strand displacement reaction.

[0079] The specific steps are as follows: 1. Plasmid Construction: The structural file of the membrane protein SLC7A5 was downloaded from the AlphaFold protein structure database. A machine learning model was used to screen for potential FSY integration sites in SLC7A5, simulating that the 220th amino acid in the SLC7A5 gene coding sequence (CDS) was the optimal insertion site for fluofenzyme-L-tyrosine (FSY). The gene encoded at position 220 was mutated to the TAG codon. The expression plasmid pcDNA3.1(+)_SLC7A5-220TAG_-C-eGFP was constructed by inserting the HindIII / NotI multiple cloning site into the pcDNA3.1(+) vector. The C-terminus of the vector contained the eGFP signal sequence. The expression plasmid pcDNA3.1(+)_SLC7A5-220TAG_-C-eGFP was synthesized by Nanjing GenScript Biotech Co., Ltd. All plasmids used in the experiment were purchased from Nanjing GenScript Biotech.

[0080] 2. Cell preparation: HEK293T cells (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were prepared at a rate of 2×10⁶ cells / year. 6 The cells were seeded at a density of 1 cell per well in six-well plates and pre-cultured in DMEM high-glucose medium containing 10% FBS for 24 hours in a 37°C, 5% CO2 incubator until the cell confluence reached 70%-80%.

[0081] 3. Transfection Complex Preparation: Take 2 μL of Lipofectamine® 2000 (Invitrogen) transfection reagent and add it to 48 μL of serum-free Opti-MEM® medium (Gibco). Gently mix and let stand for 2 min to obtain the liposome dilution. Dissolve 0.8 μg of pcDNA3.1(+)_SLC7A5-220TAG_-C-eGFP plasmid and 0.8 μg of pNEU-FSYRS plasmid (a mutant pyrrolidone-lysyl-tRNA synthetase FSYRS that specifically recognizes FSY, provided by Nanjing Genscript Biotech Co., Ltd.) in 50 μL of Opti-MEM® medium respectively and let stand at room temperature for 2 min to obtain the plasmid dilution. Slowly add the plasmid dilution to the liposome dilution, gently mix, and incubate at room temperature for 20 min to form the plasmid DNA-Lipofectamine transfection complex.

[0082] 4. 293T cell transfection and expression: The transfection complex was added dropwise to a six-well plate (an equal volume of Opti-MEM® medium was added to the blank control group). After culturing at 37°C and 5% CO2 for 8 h, the transfection mixture was removed. Fluorosulfonic acid-L-tyrosine (FSY, MCE) was weighed and dissolved in ddH2O to prepare a 100 mM solution. 2 mL of DMEM complete medium (Gibco) containing 100 mM fluorosulfonic acid-L-tyrosine (5 μL) was added to each well of the six-well plate, and the plate was cultured at 37°C and 5% CO2 for 48 h to express the FSY-modified SLC7A5 membrane protein.

[0083] 5. Adding allosteric DNA sequences: Wash the transfected 293T cells twice with pre-chilled PBS (pH 7.4) to thoroughly remove residual culture medium from the cell surface. Add the first pair of Cy3-S1S2-peptides (prepared in Example 1, 2.5 μM, 500 μL, PBS) to each well and incubate at room temperature in the dark for 30 min; discard the solution and wash once with PBS. Then add the second pair of TYR-S3S4 (prepared in Example 3, 2.5 μM, 500 μL, PBS), incubate at room temperature for 30 min, and wash once more with PBS. Add ribonuclease H (5 U / μL, 500 μL) solution to each well and incubate at room temperature for 30 min.

[0084] RNA S4 strand was degraded by ribonuclease H. The released S3 single strand then underwent a DNA strand substitution reaction with the S1S2 semi-complementary DNA double strand on the membrane protein to form an S2S3 fully complementary DNA double strand, releasing the S1 tag strand. Untransfected 293T cells were used as the control group (subsequent experimental procedures were the same, with the addition of allosteric DNA sequences).

[0085] 6. Gel Imaging: Cell supernatant solutions (to verify the target of drug TYR) were collected from each well of a six-well plate after the reaction with allosteric DNA sequences and subjected to 15% PAGE electrophoresis (Cy3-S1 as a control). Electrophoresis conditions were 120V for 120 min, followed by staining with GoldView™ nucleic acid dye in the dark for 30 min, and imaging was performed using the Bio-Rad imaging system.

[0086] The gel electrophoresis characterization results of in situ membrane protein target screening based on conformational DNA are shown below. Figure 5 The figure shows the migration of reaction products at different stages in the gel, thus verifying the effectiveness of conformational DNA in membrane protein screening.

[0087] 7. Screening of other membrane proteins: For other membrane proteins, such as SLC1A5, SLC38A1, SLC38A2 and ABCC4, the operation steps are the same as those for SLC7A5 membrane protein screening, except that the plasmid construction sequence is different.

[0088] All plasmids used in the experiment were purchased from Nanjing GenScript Biotech.

[0089] SLC1A5: The gene encoding fluorosulfonic acid-L-tyrosine was modified at amino acid position 217 of the membrane protein SLC1A5 and inserted into the pcDNA3.1(+) vector using the HindIII / NotI multiple cloning site to construct the expression plasmid pcDNA3.1(+)_SLC1A5-217TAG_-C-eGFP.

[0090] SLC38A1: The gene encoding fluorosulfonic acid-L-tyrosine was modified at amino acid position 256 of the membrane protein SLC38A1 and inserted into the pcDNA3.1(+) vector using the HindIII / NotI multiple cloning site to construct the expression plasmid pcDNA3.1(+)_SLC38A1-256TAG_-C-eGFP.

[0091] SLC38A2: The gene encoding fluorosulfonic acid-L-tyrosine was modified at amino acid position 272 of the membrane protein SLC38A2 and inserted into the pcDNA3.1(+) vector using the HindIII / NotI multiple cloning site to construct the expression plasmid pcDNA3.1(+)_SLC38A2-272TAG_-C-eGFP.

[0092] ABCC4: The gene encoding fluorosulfonic acid-L-tyrosine was modified at amino acid position 752 of the membrane protein ABCC4 and inserted into the pcDNA3.1(+) vector using the HindIII / NotI multiple cloning site to construct the expression plasmid pcDNA3.1(+)_ABCC4-752TAG_-C-eGFP.

[0093] Gel imaging results as follows Figure 5 As shown, the SLC7A5 inhibitor Diiodo-Tyr (TYR) specifically interacts only with the SLC7A5 engineered membrane protein, triggering DNA conformational changes and releasing the Cy3-S1 tag (with the same Cy3-S1 sequence position as the control group). No bands were found in other membrane protein groups expressing FSY modification, proving that this method can successfully screen for specific targets.

[0094] Example 6: Sequencing results of in situ screening of cell membrane protein targets based on conformational DNA. Building upon Example 6, this example further validates the specific sequence of the gel imaging bands through sequencing. Specifically, PCR amplification is performed on the S1 tag released by the drug TYR targeting the SLC7A5 membrane protein.

[0095] DNA was recovered and purified from the gel strips using the EZ-10 column DNA PAGE gel recovery kit (Sangon Biotech (Shanghai) Co., Ltd.), and then amplified using PCR. The reaction system is as follows: 4 μL of recovered DNA sample, 0.8 μL of forward and reverse primers (10 μM), 5 μL of 2 × Phanta Max Master Mix, and 0.2 μL of DEPC H2O; The sequence information of the upstream and downstream primers is as follows (SEQ ID NO.7-16).

[0096] SEQ ID NO.7: SLC7A5-F: 5'-TCTTCTAACCTCTCACTCGTCT-3'; SEQ ID NO.8: SLC7A5-R: 5'-GATAAGGTTTGCCGGAGGACTTAA-3'; SEQ ID NO.9: SLC1A5-F: 5'-CCACGTAGGCACAATTTTTACA-3'; SEQ ID NO.10: SLC1A5-R: 5'-GATAAGGTTTGCCGGAGGACTTAA-3'; SEQ ID NO.11: SLC38A1-F: 5'-TGTATTATCTAGTCACTCGCCTC-3'; SEQ ID NO.12: SLC38A1-R: 5'-GATAAGGTTTGCCGGAGGACTTAA-3'; SEQ ID NO.13: SLC38A2-F: 5'-ACGCTAGTAGTCACAAATCCC-3'; SEQ ID NO.14: SLC38A2-R: 5'-GATAAGGTTTGCCGGAGGACTTAA-3'; SEQ ID NO.15: ABCC4-F: 5'-ACGCTAGTAGTCACAAATCCC-3'; SEQ ID NO.16: ABCC4-R: 5'-GATAAGGTTTGCCGGAGGACTTAA-3'.

[0097] The reaction procedure is as follows: ① Pre-denaturation: 95℃ for 5 minutes; ② Denaturation, 95℃ for 15 seconds; ③ Annealing, 52℃, 15sec; ④ Extend at 72℃ for 30 seconds; repeat steps ② to ④ for a total of 45 cycles; finally extend at 72℃ for 5 minutes.

[0098] The amplified products were then used for sequencing, and the Sanger sequencing results are as follows: Figure 6 As shown in the figure, the DNA sequencing results after PCR amplification and the corresponding membrane protein target information are displayed.

[0099] The results showed that the recovered gel strip sequence was consistent with the known designed DNA tag chain encoding SLC7A5, successfully proving that the method of the present invention based on DNA tag allosteric strategy can screen out the specific target of TYR drugs as SLC7A5 in living cells in situ, and further verifying the feasibility of the method.

[0100] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for screening in situ drug membrane protein targets based on allosteric DNA, characterized in that, Includes the following steps: (1) Construct two pairs of functionalized nucleic acid structures for membrane protein tag release. The first pair is a S1S2 semi-complementary DNA double strand, in which the S1 strand is the membrane protein tag strand and the S2 strand is the functional strand coupled to the membrane protein ligand. The two form a dynamic and reversible double strand structure through semi-complementary pairing. The second pair is a S3S4 DNA-RNA hybrid double strand, in which the S3 strand is coupled to the candidate drug and the S4 strand is the RNA strand complementary to the S3 strand. (2) In living cells, non-natural amino acids are introduced into specific sites of target membrane proteins through genetic code expansion technology (GCE), and bioorthogonal reactions occur to form modifiable sites; (3) Using click chemistry, the S2 strand in the S1S2 semi-complementary DNA double-stranded structure is covalently coupled to the target membrane protein to achieve the labeling of the membrane protein; (4) Introduce the S3S4 DNA-RNA hybrid double-stranded structure into the living cell environment. When the candidate drug coupled with the S3 chain binds to the target membrane protein, it induces ribonuclease to mediate the cleavage of the S4 chain, so that the S3S4 structure becomes an S3 single strand. (5) The S3 single strand then undergoes a DNA strand substitution reaction with the S1S2 semi-complementary DNA double strand on the membrane protein to form the S2S3 fully complementary DNA double strand, releasing the S1 tag strand. (6) The released S1 tag chain is amplified by PCR and the tag sequence is obtained by Sanger / high-throughput sequencing. The target membrane protein is identified and analyzed based on the sequence information.

2. The screening method according to claim 1, characterized in that, In step (1), the S1 chain includes a partially complementary region and a coding region; the coding region sequence is a specific DNA sequence encoding a membrane protein, which varies depending on the membrane protein. The S1 strand DNA sequence is 80–110 bases long; and / or, the S2 strand DNA is 40–60 bases long; and / or, the S3 strand DNA is 50–70 bases long; and / or, the S4 strand RNA is 30–50 bases long; wherein the complementary base pairing length of the S1 and S2 strands is less than the complementary base pairing length of the S2 and S3 strands. The 5' end modification of the S2 chain with a functional group coupled to a membrane protein ligand includes any one of amino, dibenzocyclooctylene, thiol, and maleimide. The 3' end of the S3 chain is modified with a functional group that is coupled to the candidate drug, including any one of amino, dibenzocyclooctylene, mercapto, and maleimide.

3. The screening method according to claim 1, characterized in that, The S3 chain is connected to a coupling group, which is modified at the inactive site of the candidate drug with one of azide, maleimide, NHS ester, or thiol. The coupling group in the S3 chain is then bound to the candidate drug molecule via a click chemistry reaction.

4. The screening method according to claim 1, characterized in that, Includes the following steps: (1) A fluorescent group is modified at the 3' end of the S1 chain to form a "fluorescent group-S1 chain"; A membrane protein ligand is coupled to the 5' end of the S2 chain to form a "membrane protein ligand-S2 chain"; A dynamically reversible S1S2 semi-complementary DNA double strand is formed through the complementary pairing segment between the "fluorescent group-S1 chain" and the "membrane protein ligand-S2 chain". (2) The candidate drug is coupled to the 3' end of the S3 chain to form a "drug-S3 chain"; the "drug-S3 chain" is complementary to the RNA chain S4 to form an S3S4 DNA-RNA hybrid double strand; (3) In living cells, non-natural amino acids are introduced into specific sites of target membrane proteins using GCE technology to stably express non-natural amino acid-modified membrane proteins; (4) Cells that stably express non-natural amino acid-modified membrane proteins are co-incubated with S1S2 semi-complementary DNA double strands, so that they are anchored to the membrane surface through covalent binding between membrane protein ligands and special reactive functional groups on non-natural amino acids. (5) Introduce the S3S4 DNA-RNA hybrid double-stranded structure into the living cell environment. When the candidate drug coupled to the S3 chain binds to the target membrane protein, add ribonuclease to degrade the RNA chain S4, triggering the DNA chain replacement reaction, so that the "membrane protein ligand-S2 chain" and the "drug-S3 chain" are completely complementary and pair, thereby releasing the "fluorescent group-S1 chain". (6) The "fluorescent group-S1 chain" was analyzed by PCR amplification and Sanger / high-throughput sequencing, and the membrane protein target information was reverse mapped.

5. The screening method according to claim 4, characterized in that, The nucleotide sequence of the "fluorescent group-S1 chain" and the complementary pairing region of the "membrane protein ligand-S2 chain" contain 20-35 bp of complementary sequence; the complementary pairing region of the "membrane protein ligand-S2 chain" and the "drug-S3 chain" contains 40-60 bp of complementary sequence.

6. The screening method according to claim 1 or 4, characterized in that, The S1 chain is modified with the fluorescent group Cy3, and its sequence is shown in SEQ ID NO.1; the S2 chain is a DNA chain with a DBCO group modified at the 5' end, and its sequence is shown in SEQ ID NO.3; the S3 chain is modified with the DBCO group, and its sequence is shown in SEQ ID NO.5; the sequence of the S4 chain is shown in SEQ ID NO.

6.

7. The screening method according to claim 1, characterized in that, The two pairs of functionalized nucleic acid structures were constructed using a thermal annealing procedure, which is as follows: ① Reaction at 95℃ for 5 minutes; ② Reaction at 4℃ for 30 minutes.

8. The screening method according to claim 1, characterized in that, In step (3), the target membrane protein is one or more of the human cell surface membrane proteins that have been modified with non-natural amino acids.

9. The screening method according to claim 1 or 4, characterized in that, The polymerase used in the PCR amplification reaction includes one of 2×Taq plus PCR Master Mix, Platinum™ SuperFi II PCR Master Mix, or 2×PhantaMax Master Mix, and the DNA sequence length of the upstream and downstream primers is 19–22 bases.

10. The screening method according to claim 9, characterized in that, The PCR amplification reaction program is as follows: ① Pre-denaturation, 95℃ for 5 min; ② Denaturation, 95℃ for 15 sec; ③ Annealing, 52℃ for 15 sec; ④ Extension, 72℃ for 30 sec; Repeat steps ② to ④ for a total of 45 to 50 cycles; Finally, extend at 72℃ for 5 to 10 min.

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