CoolMPS antibody mutants that specifically bind to cytosine deoxynucleotides with blocking modifications and their applications

Abstract

This invention discloses a CoolMPS antibody mutant that specifically binds to a blocking-modified cytosine deoxynucleotide and its applications. The invention provides an antibody (referred to as an antibody mutant or modified antibody) that binds to a target, obtained by mutating the variable region of the heavy chain of the antibody to be modified; the mutation is located at: amino acid residue 2 of CDR1; amino acid residue 2 of CDR2; amino acid residue 15 of FR1; amino acid residue 6 of FR3; amino acid residue 21 of FR3; amino acid residue 30 of FR3; amino acid residue 38 of FR3; the target is a blocking-modified cytosine deoxynucleotide. The antibody provided by this invention can be used in basic scientific research, biosensor technology, gene sequencing, and other fields, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and high-throughput sequencing, and relates to CoolMPS antibody mutants that specifically bind to cytosine deoxynucleotides with blocking modifications and their applications. Background Technology

[0002] Massively parallel sequencing (MPS) provides billions of reads on DNA nanoarrays at a relatively low cost, enabling a wide range of genomic applications. Further improvements in read length, sequence quality, and cost reduction will make more affordable, accurate, and comprehensive health monitoring tests possible. In modern synthetic sequencing, the most efficient MPS uses fluorescently labeled reversible terminated nucleotides (RTs), but these nucleotides have the following limitations: ① high manufacturing costs and difficulty in incorporation; ② a portion of the fluorescent linker (scar) remains on the nucleotide after cleavage, interfering with subsequent sequencing cycles.

[0003] In 2020, BGI Genomics developed a novel sequencing chemistry method, CoolMPS, which uses unlabeled reversible terminators (RTs) and naturally occurring base-specific fluorescently labeled antibodies to successfully and effectively improve sequencing signal strength, enhancing readability and accuracy. In each sequencing cycle of CoolMPS, fluorescently labeled antibodies based on 3' blocking are used to merge unlabeled reversible terminators and determine the bases. Removing the bound antibody and the 3' blocking site on the nucleotide sugar group restores the nucleotide to its natural, scar-free nucleotide state. This return to natural nucleotides allows for further DNA strand extension in new sequencing cycles without interference from previous cycles. Furthermore, unlabeled reversible terminators (RTs) are easier and less expensive to manufacture, and they can be added more efficiently. Another advantage of CoolMPS is that the antibody can carry multiple molecules (e.g., 2-5) of the same dye, significantly increasing the sequencing signal compared to a single dye on standard labeled RTs.

[0004] Currently, the antibodies used in the CoolMPS sequencing method are obtained through in vivo immunization and ELISA screening. Specifically, to generate the desired antibody, a blocking nucleotide with a blocking group is linked to an N-hydroxysuccinimide group via a monophosphate ester, which is then linked to the KLH protein for immunization of animals every two weeks. Serum is collected from the animals over three months and screened using ELISA to determine the immune response. This method has low yield, is time-consuming and costly, and offers limited improvement in sequencing accuracy. Summary of the Invention

[0005] This invention aims to design high-affinity antibodies that specifically recognize cytosine deoxynucleotides with blocking modifications. The inventors of this invention modified existing CoolMPS sequencing antibodies targeting cytosine deoxynucleotides with blocking modifications, using artificial intelligence methods and molecular dynamics simulations to design antibodies with better affinity and specific recognition, enabling longer and more accurate sequencing reads in CoolMPS sequencing. The antibodies provided by this invention are obtained by mutation of the original antibody; therefore, the antibodies provided by this invention are also called antibody mutants or modified antibodies.

[0006] The present invention provides an antibody that binds to a target (referred to as an antibody mutant or modified antibody), characterized in that: the antibody is obtained by mutating the variable region of the heavy chain of the antibody to be modified;

[0007] The variable region of the heavy chain of the antibody to be modified includes three complementary determinant regions and four frame regions; the three complementary determinant regions are H-CDR1, H-CDR2, and H-CDR3; the four frame regions are H-FR1, H-FR2, H-FR3, and H-FR4; the amino acid sequence of H-CDR1 is SEQ ID NO: 1 or has more than 80% identity with SEQ ID NO: 1, the amino acid sequence of H-CDR2 is SEQ ID NO: 2 or has more than 80% identity with SEQ ID NO: 2, the amino acid sequence of H-FR1 is SEQ ID NO: 4 or has more than 80% identity with SEQ ID NO: 4, and the amino acid sequence of H-FR3 is SEQ ID NO: 6 or has more than 80% identity with SEQ ID NO: 6;

[0008] The mutations are located in the following (a1) and / or (a2) and / or (a3) ​​and / or (a4) and / or (a5) and / or (a6) and / or (a7):

[0009] (a1) The second amino acid residue of H-CDR1;

[0010] (a2) The second amino acid residue of H-CDR2;

[0011] (a3) The 15th amino acid residue of H-FR1;

[0012] (a4) The 6th amino acid residue of H-FR3;

[0013] (a5) The 21st amino acid residue of H-FR3;

[0014] (a6) The 30th amino acid residue of H-FR3;

[0015] (a7) The 38th amino acid residue of H-FR3;

[0016] The target is a cytosine deoxynucleotide with blocking modification.

[0017] The mutations are as follows: (b1) and / or (b2) and / or (b3) and / or (b4) and / or (b5) and / or (b6) and / or (b7):

[0018] (b1) The second amino acid residue of H-CDR1 is mutated from F to Y;

[0019] (b2) The second amino acid residue of H-CDR2 is mutated from I to Y or S;

[0020] (b3) The 15th amino acid residue of H-FR1 is mutated from E to G;

[0021] (b4) The 6th amino acid residue W of H-FR3 is mutated to P, F, or H;

[0022] (b5) The 21st amino acid residue of H-FR3 is mutated from M to T;

[0023] (b6) The 30th amino acid residue of H-FR3 is mutated from D to S;

[0024] (b7) The 38th amino acid residue of H-FR3 is mutated from S to Y.

[0025] The amino acid sequence of H-CDR3 is SEQ ID NO:3 or has more than 80% identity with SEQ ID NO:3.

[0026] The amino acid sequence of H-FR2 is SEQ ID NO:5, which has more than 80% identity with SEQ ID NO:5.

[0027] The amino acid sequence of H-FR4 is SEQ ID NO:7, which has more than 80% identity with SEQ ID NO:7.

[0028] Specifically, the variable region of the heavy chain of the antibody to be modified includes the segment shown in positions 1-120 of SEQ ID NO: 8. Specifically, the variable region of the heavy chain of the antibody to be modified includes a segment with more than 80% identity to positions 1-120 of SEQ ID NO: 8. Specifically, the variable region of the heavy chain of the antibody to be modified is as shown in positions 1-120 of SEQ ID NO: 8. Specifically, the variable region of the heavy chain of the antibody to be modified has more than 80% identity to positions 1-120 of SEQ ID NO: 8.

[0029] Specifically, the constant region of the heavy chain of the antibody to be modified includes the segment shown in positions 121-442 of SEQ ID NO: 8. Specifically, the constant region of the heavy chain of the antibody to be modified includes a segment with more than 80% identity to positions 121-442 of SEQ ID NO: 8. Specifically, the constant region of the heavy chain of the antibody to be modified is as shown in positions 121-442 of SEQ ID NO: 8. Specifically, the constant region of the heavy chain of the antibody to be modified has more than 80% identity to positions 121-442 of SEQ ID NO: 8.

[0030] Specifically, the heavy chain of the antibody to be modified includes the segment shown in SEQ ID NO: 8. Specifically, the heavy chain of the antibody to be modified includes a segment with more than 80% identity to SEQ ID NO: 8. Specifically, the heavy chain of the antibody to be modified is as shown in SEQ ID NO: 8. Specifically, the heavy chain of the antibody to be modified has more than 80% identity to SEQ ID NO: 8.

[0031] The variable region of the light chain of the antibody to be modified includes three complementary determinant regions; the three complementary determinant regions are L-CDR1, L-CDR2, and L-CDR3. L-CDR1 is as shown in positions 26-38 of SEQ ID NO: 10 or has more than 80% identity with positions 26-38 of SEQ ID NO: 10. L-CDR2 is as shown in positions 54-60 of SEQ ID NO: 10 or has more than 80% identity with positions 54-60 of SEQ ID NO: 10. L-CDR3 is as shown in positions 93-101 of SEQ ID NO: 10 or has more than 80% identity with positions 93-101 of SEQ ID NO: 10.

[0032] The variable region of the light chain of the antibody to be modified includes four frame regions, namely L-FR1, L-FR2, L-FR3, and L-FR4. L-FR1 is as shown in positions 1-25 of SEQ ID NO: 10 or has more than 80% identity with positions 1-25 of SEQ ID NO: 10. L-FR2 is as shown in positions 39-53 of SEQ ID NO: 10 or has more than 80% identity with positions 39-53 of SEQ ID NO: 10. L-FR3 is as shown in positions 61-92 of SEQ ID NO: 10 or has more than 80% identity with positions 61-92 of SEQ ID NO: 10. L-FR4 is as shown in positions 102-111 of SEQ ID NO: 10 or has more than 80% identity with positions 102-111 of SEQ ID NO: 10.

[0033] Specifically, the variable region of the light chain of the antibody to be modified includes the segment shown in positions 1-111 of SEQ ID NO: 10. Specifically, the variable region of the light chain of the antibody to be modified includes a segment with more than 80% identity to positions 1-111 of SEQ ID NO: 10. Specifically, the variable region of the light chain of the antibody to be modified is as shown in positions 1-111 of SEQ ID NO: 10. Specifically, the variable region of the light chain of the antibody to be modified has more than 80% identity to positions 1-111 of SEQ ID NO: 10.

[0034] Specifically, the constant region of the light chain of the antibody to be modified includes the segment shown in positions 112-215 of SEQ ID NO: 10. Specifically, the constant region of the light chain of the antibody to be modified includes a segment with more than 80% identity to positions 112-215 of SEQ ID NO: 10. Specifically, the constant region of the light chain of the antibody to be modified is as shown in positions 112-215 of SEQ ID NO: 10. Specifically, the constant region of the light chain of the antibody to be modified has more than 80% identity to positions 112-215 of SEQ ID NO: 10.

[0035] Specifically, the light chain of the antibody to be modified includes the segment shown in SEQ ID NO: 10. Specifically, the light chain of the antibody to be modified includes a segment with more than 80% identity to SEQ ID NO: 10. Specifically, the light chain of the antibody to be modified is as shown in SEQ ID NO: 10. Specifically, the light chain of the antibody to be modified has more than 80% identity to SEQ ID NO: 10.

[0036] This invention does not modify the light chain of the antibody to be modified.

[0037] Therefore, the light chain of the antibody mutant provided by the present invention is consistent with the light chain of the antibody to be modified.

[0038] The antibody mutants provided by this invention can be prepared using any existing method, including but not limited to: co-introducing a plasmid expressing the light chain and a plasmid expressing the heavy chain into a host cell, followed by cell culture to obtain the antibody mutant. The cells include, but are not limited to, CHO cells or HEK293 cells. Methods for introducing the plasmids into the host include, but are not limited to, electroporation, liposome-mediated methods, and FuGENE6 transfection reagent-mediated methods.

[0039] This invention also protects the antigen-binding fragment of any of the antibody mutants described above.

[0040] The naming system for the CDR is Chothia.

[0041] This invention also protects antibody-related biomaterials;

[0042] The antibody-related biomaterials are (c1), (c2), (c3), (c4), or (c5) as follows:

[0043] (c1) A polynucleotide encoding the antibody mutant;

[0044] (c2) An expression vector having the polynucleotide described in (c1);

[0045] (c3) A recombinant cell expressing the antibody mutant;

[0046] (c4) A recombinant cell having the polynucleotide described in (c1);

[0047] (c5) A recombinant cell having the expression vector described in (c2).

[0048] Optionally, the expression vector is a eukaryotic expression vector.

[0049] Optionally, the recombinant cells are obtained by introducing the expression vector into a host cell.

[0050] This invention also protects biomaterials related to antigen-binding fragments;

[0051] The antigen-binding fragment-related biological material is as follows (d1), (d2), (d3), (d4), or (d5):

[0052] (d1) A polynucleotide encoding the antigen-binding fragment;

[0053] (d2) An expression vector having the polynucleotide described in (d1);

[0054] (d3) A recombinant cell that expresses the antigen-binding fragment;

[0055] (d4) A recombinant cell having the polynucleotide described in (d1);

[0056] (d5) A recombinant cell having the expression vector described in (d2).

[0057] Optionally, the expression vector is a eukaryotic expression vector.

[0058] Optionally, the recombinant cells are obtained by introducing the expression vector into a host cell.

[0059] This invention also protects a complex, which is a conjugate obtained by chemically linking substance A and substance B; wherein substance A is any of the antibody mutants or antigen-binding fragments described above; and substance B is a small molecule compound, a macromolecule compound, or a polymer.

[0060] Optionally, the small molecule compound is a fluorescent dye.

[0061] Optionally, the macromolecular compound is a fluorescent dye.

[0062] Optionally, the polymer is a fluorescent dye.

[0063] The present invention also protects the use of any of the antibody mutants or antigen-binding fragments described above, as follows (e1) or (e2) or (e3) or (e4):

[0064] (e1) Specific binding to the target;

[0065] (e2) Prepare reagents or kits for the specific binding of targets;

[0066] (e3) Nucleic acid sequencing;

[0067] (e4) Prepare reagents or kits for nucleic acid sequencing;

[0068] The target is: cytosine deoxynucleotide with blocking modification;

[0069] Optionally, the nucleic acid sequencing is CoolMPS sequencing.

[0070] The present invention also provides a method for detecting a target, comprising the following steps: detecting whether the target is present in the analyte by contacting the analyte with a label; wherein the target is a cytosine deoxynucleotide with blocking modification; and the label is obtained by labeling any of the antibody mutants or antigen-binding fragments described above.

[0071] Specifically, the marking is a fluorescent marker (fluorescent group marker), such as AF532, EF660, etc.

[0072] The present invention also provides a sequencing method, comprising the following steps:

[0073] The nucleic acid to be tested, four cold dNTPs and DNA polymerase were mixed and then amplified under conditions suitable for nucleic acid amplification.

[0074] The amplified product after the amplification process was combined with four markers and subjected to fluorescence signal detection processing.

[0075] Based on the fluorescence signal obtained from the fluorescence signal detection and processing, the nucleic acid sequence of the nucleic acid to be tested is determined;

[0076] The four markers are: marker 1, obtained by labeling an antibody or antigen-binding fragment that binds to a cytosine deoxynucleotide with a blocking modification; marker 2, obtained by labeling an antibody or antigen-binding fragment that binds to an adenine deoxynucleotide with a blocking modification; marker 3, obtained by labeling an antibody or antigen-binding fragment that binds to a guanine deoxynucleotide with a blocking modification; and marker 4, obtained by labeling an antibody or antigen-binding fragment that binds to a thymine deoxynucleotide with a blocking modification.

[0077] The antibody that binds to the cytosine deoxynucleotide with blocking modification is any of the antibody mutants described above; the antigen-binding fragment that binds to the cytosine deoxynucleotide with blocking modification is any of the antigen-binding fragments described above.

[0078] Specifically, marker 1 is obtained by fluorescently labeling an antibody or antigen-binding fragment that binds to a cytosine deoxynucleotide with a blocking modification. Marker 2 is obtained by fluorescently labeling an antibody or antigen-binding fragment that binds to an adenine deoxynucleotide with a blocking modification. Marker 3 is obtained by fluorescently labeling an antibody or antigen-binding fragment that binds to a guanine deoxynucleotide with a blocking modification. Marker 4 is obtained by fluorescently labeling an antibody or antigen-binding fragment that binds to a thymine deoxynucleotide with a blocking modification. Specifically, the four markers have different fluorescent labels.

[0079] Cold dNTPs refer to dNTPs without labeled fluorescent groups.

[0080] The four types of cold dNTPs refer to dCTP, dATP, dGTP, and dTTP.

[0081] Optionally, the nucleic acid sequencing is CoolMPS sequencing.

[0082] The present invention also provides a sequencing kit, characterized in that it comprises any of the antibody mutants described above, any of the antigen-binding fragments described above, any of the biological materials described above, or any of the complexes described above.

[0083] Optionally, the kit further comprises the following components:

[0084] Antibodies or antigen-binding fragments thereof that bind to adenine deoxynucleotides with blocking modifications;

[0085] Antibodies or antigen-binding fragments thereof that bind to guanine deoxynucleotides with blocking modifications;

[0086] Antibodies or antigen-binding fragments thereof that bind to thymine deoxynucleotides with blocking modifications.

[0087] Optionally, the kit further includes at least one component selected from DNA polymerase, DNA polymerase buffer, and cold dNTPs.

[0088] The present invention also provides a nucleotide localization method, characterized by comprising the following steps:

[0089] The primers, the nucleic acid sample to be tested, and any of the complexes described above are mixed and processed.

[0090] The product of the above-mentioned mixing process is subjected to fluorescence signal detection processing;

[0091] Based on the fluorescence signal obtained from the fluorescence signal detection and processing, the location of the target in the nucleic acid sample to be tested is determined; the target is a cytosine deoxynucleotide with blocking modification.

[0092] Optionally, the above-mentioned 80% or more identity can be 85% or more identity, or 90% or more identity, or 91% or more identity, or 92% or more identity, or 93% or more identity, or 94% or more identity, or 95% or more identity, or 96% or more identity, or 97% or more identity, or 98% or more identity, or 99% or more identity.

[0093] Specifically, any of the targets mentioned above can be: independent cytosine deoxynucleotides with blocking modifications.

[0094] Specifically, any of the targets mentioned above can be: cytosine deoxynucleotides with blocking modifications located at the 3' end of a DNA molecule.

[0095] Specifically, the DNA molecule is obtained by sequentially hybridizing and extending a template strand and an amplification strand; the template strand is a single-stranded DNA molecule composed of n nucleotides, and the amplification strand is a single-stranded DNA molecule composed of nx nucleotides; the template strand and the amplification strand are inversely complementary. n is a natural number greater than 6. x is 1, 2, 3, 4, or 5. Specifically, the extension is a single nucleotide extension. Specifically, the extension refers to: according to the principle of inverse complementarity with the template strand, the amplification strand extends a cytosine deoxynucleotide with a blocking modification at its 3' end. Specifically, the 5' end of the amplification strand has a biotin modification.

[0096] Specifically, the blocking groups of the blocking modification include, but are not limited to: O-Azidomethyl, Amino, allyl, Thiol, Phosphate, Azido, or Methyl ester.

[0097] According to specific embodiments of the present invention, the antibody mutants provided by the present invention can effectively recognize natural dNTP substrates, exhibiting higher signal intensity and higher signal-to-noise ratio in sequencing scenarios. Especially in high-density nanoarrays, even with low template copy numbers of DNBs, sufficient signals can be generated, resulting in more accurate sequencing results and longer read lengths.

[0098] According to some specific embodiments of the present invention, the antibody mutants provided by the present invention can be widely used in genome sequencing, diagnostic sequencing, multiplex nucleic acid amplification, therapeutic antibody development, synthetic biology, nucleic acid therapy, DNA assembly, and DNA data storage.

[0099] Compared to the original antibody, the antibody mutant provided by this invention exhibits significantly enhanced specificity and affinity for the substrate, and lower crosstalk, meaning that its application in CoolMPS sequencing can improve sequencing accuracy. The antibody mutant provided by this invention has broad application prospects in basic scientific research, biosensor technology, and gene sequencing. Attached Figure Description

[0100] Figure 1 This is a schematic diagram (element diagram) of the recombinant plasmid.

[0101] Figure 2 This is an SDS-PAGE electrophoresis image of the antibody solution.

[0102] Figure 3 UREA-PAGE gel electrophoresis image for preparing CoolMPS antigen C.

[0103] Figure 4 SPR results for determining the affinity of the antibody for CoolMPS antigen C.

[0104] Figure 5 SPR results for determining the affinity of the antibody for CoolMPS antigen A.

[0105] Figure 6 SPR results for determining the affinity of the antibody for CoolMPS antigen T.

[0106] Figure 7SPR results for determining the affinity of the antibody for CoolMPS antigen G. Detailed Implementation

[0107] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0108] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged. In the examples, the recombinant plasmids have all been sequenced for verification.

[0109] 3'-O-Azidomethyl-dATP (specification: 100mM solution): Shenji Biotechnology Co., Ltd., product catalog number dNTP023. 3'-O-Azidomethyl-dGTP (specification: 100mM solution): Shenji Biotechnology Co., Ltd., product catalog number dNTP024. 3'-O-Azidomethyl-dCTP (specification: 100mM solution): Shenji Biotechnology Co., Ltd., product catalog number dNTP025. 3'-O-Azidomethyl-dTTP (specification: 100mM solution): Shenji Biotechnology Co., Ltd., product catalog number dNTP026. All four compounds should be diluted with ultrapure water to a concentration of 100μM before use.

[0110] Example 1: Design and Construction of CoolMPS Antibody Plasmid

[0111] The exogenous DNA molecule was inserted between the NotⅠ and XbaⅠ restriction sites of the pcDNA3.1(+) plasmid to obtain the recombinant plasmid. A map of the recombinant plasmid is shown below. Figure 1 .

[0112] When the exogenous DNA molecule is the double-stranded DNA molecule shown in SEQ ID NO: 11, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-L. The double-stranded DNA molecule shown in SEQ ID NO: 11 encodes the protein shown in SEQ ID NO: 10, and the protein is named the universal light chain.

[0113] When the exogenous DNA molecule is the double-stranded DNA molecule shown in SEQ ID NO: 9, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. WT The double-stranded DNA molecule shown in SEQ ID NO: 9 encodes the protein shown in SEQ ID NO: 8, and this protein is named ABCD_350 heavy chain.

[0114] When the exogenous DNA molecule is DNA molecule I, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. E15G Compared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference of DNA molecule I is that the nucleotides at positions 43-45 are mutated from GAG to GGA, thereby encoding an amino acid residue (i.e., amino acid residue at position 15 in SEQ ID NO: 8) that is mutated from E to G. The mutant protein obtained by mutating amino acid residue at position 15 of SEQ ID NO: 8 from E to G is named ABCS_3 heavy chain.

[0115] When the exogenous DNA molecule is DNA molecule II, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. F27Y Compared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference of DNA molecule II is that the nucleotides at positions 79-81 are mutated from TTC to TAC, thereby encoding an amino acid residue (i.e., amino acid residue at position 27 in SEQ ID NO: 8) that is mutated from F to Y. The mutant protein obtained by mutating amino acid residue at position 27 of SEQ ID NO: 8 from F to Y is named ABCS_4 heavy chain.

[0116] When the exogenous DNA molecule is DNA molecule III, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. I54Y Compared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference of DNA molecule III is that the nucleotides at positions 160-162 are mutated from ATC to TAC, thereby encoding an amino acid residue (i.e., amino acid residue at position 54 in SEQ ID NO: 8) that is mutated from I to Y. The mutant protein obtained by mutating amino acid residue at position 54 of SEQ ID NO: 8 from I to Y is named ABCS_31 heavy chain.

[0117] When the exogenous DNA molecule is DNA molecule IV, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. I54SCompared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference of DNA molecule IV is that the nucleotides at positions 160-162 are mutated from ATC to TCC, thereby encoding an amino acid residue (i.e., amino acid residue at position 54 in SEQ ID NO: 8) that is mutated from I to S. The mutant protein obtained by mutating amino acid residue at position 54 of SEQ ID NO: 8 from I to S is named ABCS_33 heavy chain.

[0118] When the exogenous DNA molecule is DNA molecule V, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. W64P Compared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference in DNA molecule V is that the nucleotides at positions 190-192 are mutated from TGG to CCT, thereby encoding an amino acid residue (i.e., amino acid residue at position 64 in SEQ ID NO: 8) that is mutated from W to P. The mutant protein obtained by mutating amino acid residue at position 64 of SEQ ID NO: 8 from W to P is named ABCS_48 heavy chain.

[0119] When the exogenous DNA molecule is DNA molecule VI, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. W64F Compared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference in DNA molecule VI is that the nucleotides at positions 190-192 are mutated from TGG to TTC, thereby encoding an amino acid residue (i.e., amino acid residue at position 64 in SEQ ID NO: 8) that is mutated from W to F. The mutant protein obtained by mutating amino acid residue at position 64 of SEQ ID NO: 8 from W to F is named ABCS_59 heavy chain.

[0120] When the exogenous DNA molecule is DNA molecule VII, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. W64H Compared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference in DNA molecule VII is that the nucleotides at positions 190-192 are mutated from TGG to CAC, thereby encoding an amino acid residue (i.e., amino acid residue at position 64 in SEQ ID NO: 8) that is mutated from W to H. The mutant protein obtained by mutating amino acid residue at position 64 of SEQ ID NO: 8 from W to H is named ABCS_63 heavy chain.

[0121] When the exogenous DNA molecule is DNA molecule VIII, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. M79TCompared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference in DNA molecule VIII is that the nucleotides at positions 235-237 are mutated from ATG to ACC, thereby encoding an amino acid residue (i.e., amino acid residue at position 79 in SEQ ID NO: 8) that is mutated from M to T. The mutant protein obtained by mutating amino acid residue at position 79 of SEQ ID NO: 8 from M to T is named ABCS_72 heavy chain.

[0122] When the exogenous DNA molecule is DNA molecule IX, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. D88S Compared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference in DNA molecule IX is that the nucleotides at positions 262-264 are mutated from GAC to TCC, thereby encoding an amino acid residue (i.e., amino acid residue at position 88 in SEQ ID NO: 8) that is mutated from D to S. The mutant protein obtained by mutating amino acid residue at position 88 of SEQ ID NO: 8 from D to S is named ABCS_78 heavy chain.

[0123] When the exogenous DNA molecule is DNA molecule X, the resulting recombinant plasmid is named pCDNA3.1-CoolMPS-H. S96Y Compared to the double-stranded DNA molecule shown in SEQ ID NO: 9, the only difference in DNA molecule X is that the nucleotides at positions 286-288 are mutated from TCC to TAC, thereby encoding an amino acid residue (i.e., amino acid residue at position 96 in SEQ ID NO: 8) that is mutated from S to Y. The mutant protein obtained by mutating amino acid residue at position 96 of SEQ ID NO: 8 from S to Y is named ABCS_81 heavy chain.

[0124] Example 2: CoolMPS antibody expression and purification

[0125] The light chain plasmid is: pCDNA3.1-CoolMPS-L.

[0126] The heavy chain plasmids are: pCDNA3.1-CoolMPS-H WT pCDNA3.1-CoolMPS-H E15G pCDNA3.1-CoolMPS-H F27Y pCDNA3.1-CoolMPS-H I54Y pCDNA3.1-CoolMPS-H I54S pCDNA3.1-CoolMPS-H W64P pCDNA3.1-CoolMPS-H W64F pCDNA3.1-CoolMPS-HW64H pCDNA3.1-CoolMPS-H M79T pCDNA3.1-CoolMPS-H D88S or pCDNA3.1-CoolMPS-H S96Y .

[0127] 1. Using the Celestrix / SLT electroporator and following the instructions, introduce the light chain plasmid and heavy chain plasmid into CHO cells via electroporation, and then culture them in ExpiCHO expression medium at 37°C for 6-10 days.

[0128] 2. After completing step 1, centrifuge at 3000-4000g for 20-30 min, collect the supernatant, then filter using a 0.45μm filter membrane, collect the filtrate, and then perform affinity chromatography using Protein At Beads LX (Changzhou Tiandi Renhe Biotechnology Co., Ltd., product catalog number SA085100, operate according to the instructions), elute with Glycine-HCl buffer (pH 2.5-3.0, 100mM), and collect the post-column solution.

[0129] 3. Take the post-column solution obtained in step 2 and use... Antibody purity was determined by gel filtration chromatography (using PBS buffer as the mobile phase) with 200 Increase 10 / 300 GL (Cytiva, 28-9909-44). The results showed that the antibody purity was above 85%.

[0130] 4. Take the post-column solution obtained in step 2 and concentrate it using a 50 kDa concentration tube (replacing the buffer system with PBS buffer) to obtain the antibody solution (i.e., PBS buffer containing purified antibody). The antibody concentration in the solution was measured to be 0.5-3 mg / mL.

[0131] Each antibody was named according to the principle of corresponding to the heavy chain name. For example, the light chain plasmid pCDNA3.1-CoolMPS-L and the heavy chain plasmid pCDNA3.1-CoolMPS-H were used. WT The antibody obtained by performing the above steps is composed of a universal light chain and an ABCD_350 heavy chain, namely the ABCD_350 antibody, and the corresponding antibody solution is named ABCD_350 antibody solution. For example, the light chain plasmid pCDNA3.1-CoolMPS-L and the heavy chain plasmid pCDNA3.1-CoolMPS-H can be used. E15G The above steps yield an antibody composed of a universal light chain and an ABCS_3 heavy chain, known as an ABCS_3 antibody, and the corresponding antibody solution is named an ABCS_3 antibody solution. This process continues in the same manner.

[0132] 5. The SDS-PAGE electrophoresis images of the various antibody solutions prepared in step 4 are shown below. Figure 2 (In the figure, M refers to the protein marker, and NR refers to the non-reducing SDS-PAGE).

[0133] Example 3: Preparation of CoolMPS antigen

[0134] In this embodiment, CoolMPS antigen was prepared using an enzymatic synthesis technique.

[0135] I. Preparation of CoolMPS antigen C

[0136] 1. Prepare template strand and amplification strand separately.

[0137] Both the template strand and the amplification strand are single-stranded DNA molecules, and the 5' end of the amplification strand is modified with biotin.

[0138] Template chain: 5'-AAGTCGGATCGTAGCCATGTCGTTCTG-3';

[0139] Amplification chain: 5'-Biotin-CAGAACGACATGGCTACGAT-3'.

[0140] 2. Mix equal volumes of 100μM template chain solution and 100μM amplification chain solution, incubate at 80-95℃ for 5-15 minutes, and allow to cool naturally to obtain the product solution.

[0141] 3. Prepare the reaction system and then incubate at 40-50℃ for 30 minutes.

[0142] The composition of the reaction system (total volume 50 μL) is shown in Table 1.

[0143] Set up a control system: The only difference from Table 1 is that DNA polymerase is not added.

[0144] Table 1

[0145] Components Volume (μL) 3'-O-Azidomethyl-dCTP (100 μM) 1.25 The product solution obtained in step 2 1 2×PBS buffer 25 1 mg / ml DNA polymerase 5 <![CDATA[ddH2O]]> Make up to 50 μL

[0146] 4. After completing step 3, take samples for 20% UREA-PAGE gel electrophoresis. The results are shown in [the table below]. Figure 3 (The 27nt band is the template strand, and the 20nt band is the amplification strand). Due to the addition of 3'-O-Azidomethyl-dCTP, the size of the extension product is 21nt. The extension product is CoolMPS antigen C (a cytosine deoxynucleotide with a blocking modification is extended from the 3' end of the amplification strand in the hybridization strand formed by the template and amplification strands). Figure 3This indicates that by adding a blocking cytosine deoxynucleotide to the 3' end of almost all amplified strands using polymerase, high-purity CoolMPS antigen C was prepared, which can be directly used for downstream antigen chip immobilization and other detection experiments.

[0147] II. Preparation of CoolMPS Antigen A

[0148] The only difference from step one is that the amplified strand sequence is different, and 3'-O-Azidomethyl-dCTP is replaced with 3'-O-Azidomethyl-dATP.

[0149] Amplified strand (SEQ ID NO: 7): 5'-Biotin-CAGAACGACATGGCTACG-3'.

[0150] The extended product is CoolMPS antigen A (an adenine deoxynucleotide with a blocking modification is extended to the 3' end of the amplified strand in the hybridized strand formed by the template strand and the amplified strand). UREA-PAGE gel electrophoresis results show that high-purity CoolMPS antigen A was prepared and can be directly used for downstream antigen chip immobilization and other detection experiments.

[0151] III. Preparation of CoolMPS antigen T

[0152] The only difference from step one is that the amplified strand sequence is different, and 3'-O-Azidomethyl-dCTP is replaced with 3'-O-Azidomethyl-dTTP.

[0153] Amplification chain: 5'-Biotin-CAGAACGACATGGCTACGA-3'.

[0154] The extended product is CoolMPS antigen T (a thymine deoxynucleotide with a blocking modification is extended to the 3' end of the amplified strand in the hybridized strand formed by the template strand and the amplified strand). UREA-PAGE gel electrophoresis results show that high-purity CoolMPS antigen T was prepared and can be directly used for downstream antigen chip immobilization and other detection experiments.

[0155] IV. Preparation of CoolMPS Antigen G

[0156] The only difference from step one is that the amplified strand sequence is different, and 3'-O-Azidomethyl-dCTP is replaced with 3'-O-Azidomethyl-dGTP.

[0157] Amplification chain: 5'-Biotin-CAGAACGACATGGCTAC-3'.

[0158] The extended product is CoolMPS antigen G (a guanine deoxynucleotide with a blocking modification is extended to the 3' end of the amplified strand in the hybridized strand formed by the template strand and the amplified strand). UREA-PAGE gel electrophoresis results show that high-purity CoolMPS antigen G was prepared and can be directly used for downstream antigen chip immobilization and other detection experiments.

[0159] Example 4: CoolMPS Antibody Affinity Test

[0160] This embodiment uses surface plasmon resonance (SPR) technology to determine the affinity between the CoolMPS antibody mutant and CoolMPS antigen C. Ligand: CoolMPS antigen C prepared in step one of Example 3, diluted to a concentration of 20 nM with buffer before use. Analytes: Various antibodies prepared in Example 2, diluted to different concentrations with buffer before use. All buffers used in this embodiment are HBS-EP+ buffer. Regeneration solution: 10 mM Glycine-HCl buffer. The instrument used for detection is a commercial SPR instrument (InBio, Molecular Interaction Analyzer MI-S200D).

[0161] First, the ligand is immobilized on the sensor chip surface (using biotin at the 5' end of the amplification chain). Then, sample injection is performed (a solution containing the analyte is flowed through the sensor chip surface via a microfluidic system; the interaction between the ligand and the analyte causes a change in the SPR angle, which is monitored and recorded by the instrument in real time). Next, data acquisition is performed (the instrument records changes in the SPR signal during the binding and subsequent dissociation of the analyte and ligand). Then, chip regeneration is performed (the chip is cleaned with a regeneration solution to remove residual analyte, restoring the chip surface and preparing it for the next round of experiments). Finally, data analysis is performed (the SPR signals acquired during the experiment are processed by analysis software to calculate intermolecular binding affinity, kinetic parameters, etc., thus obtaining detailed information about biomolecular interactions). Relevant operating parameters are shown in Table 2.

[0162] Table 2

[0163]

[0164] See results Figure 4And Table 3 (the larger the SPR affinity signal value, the higher the affinity between the analyte and the ligand). It can be seen that CoolMPS antibodies ABCD_350, ABCS_3, ABCS_4, ABCS_31, ABCS_33, ABCS_48, ABCS_59, ABCS_63, ABCS_72, ABCS_78, and ABCS_81 all exhibited strong affinity. Among them, CoolMPS antibodies ABCS_3, ABCS_4, ABCS_31, ABCS_33, ABCS_48, ABCS_59, ABCS_63, ABCS_72, ABCS_78, and ABCS_81 showed significantly higher affinity than CoolMPS antibody ABCD_350.

[0165] Table 3

[0166]

[0167] Example 5: CoolMPS Antibody Specificity Test

[0168] This embodiment uses surface plasmon resonance (SPR) technology to determine the affinity of CoolMPS antibody mutants for CoolMPS antigen A, CoolMPS antigen T, or CoolMPS antigen G, in order to evaluate the specificity of CoolMPS antibody mutants. The method is basically the same as in Example 4. The only difference is in the ligand and positive control.

[0169] The ligands were: CoolMPS antigen A prepared in step two of Example 3, CoolMPS antigen T prepared in step three, and CoolMPS antigen G prepared in step four.

[0170] Set up a positive control for the analyte, namely, the existing CoolMPS antibodies (CoolMPS antibodies against thymine deoxynucleotides with blocking modifications, CoolMPS antibodies against adenine deoxynucleotides with blocking modifications, and CoolMPS antibodies against guanine deoxynucleotides with blocking modifications; all antibody components provided in the kit). Kit information: CoolMPS High-Throughput Sequencing Reagent Kit (MGISEQ-2000RS FCL PE100), manufactured by BGI Genomics, product catalog number 1000018238.

[0171] See results Figure 5See Table 3. CoolMPS antibody A (positive control A) shows a significant affinity for antigen A. The antibodies prepared in this invention (including CoolMPS antibodies ABCD_350, ABCS_3, ABCS_4, ABCS_31, ABCS_33, ABCS_48, ABCS_59, ABCS_63, ABCS_72, ABCS_78, and ABCS_81) show no affinity for antigen A. The results for antigens T and G are similar to those for antigen A. This indicates that CoolMPS antibody C prepared in this invention has no affinity for antigens A, T, and G, but exhibits high affinity and high specificity for antigen C.

[0172] Example 6: CoolMPS sequencing using a CoolMPS antibody mutant

[0173] This embodiment provides an exemplary method for CoolMPS sequencing using CoolMPS antibody mutants.

[0174] The sequencing reagent kit used was the CoolMPS reagent kit from BGI Genomics, along with standard balanced DNA libraries (the DNA library to be tested) and the BGI Genomics MPS platform DNBSEQ-G400.

[0175] The tested antibodies were the various antibodies prepared in Example 2.

[0176] 1. The test antibodies were fluorescently labeled to obtain individual fluorescently labeled antibodies.

[0177] The test antibody was fluorescently labeled (using the NHS crosslinking reaction to couple the antibody and the fluorescent group): The antibody (1 mg / ml) was adjusted to pH 8 in bicarbonate buffer. The NHS ester-activated fluorophore (AF532 or EF660) was diluted to 10-100 μM with anhydrous dimethyl sulfoxide. The antibody was incubated with the NHS ester dye at room temperature for 45 min, and then quenched by adding Tris buffer (pH 7.4). After labeling, the antibody was centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was then aliquoted and stored at -20 °C for later use.

[0178] II. CoolMPS sequencing using fluorescently labeled antibodies

[0179] CoolMPS sequencing was performed using an existing kit, largely following the instructions. The only difference was that the fluorescently labeled antibodies prepared in step one were used instead of the original CoolMPS antibodies in the kit that target cytosine deoxynucleotides with blocking modifications.

[0180] In each sequencing cycle of CoolMPS sequencing, dNTPs with blocking groups at the 3' end and no fluorescent labeling on the bases (referred to as cold dNTPs) are used. Cold dNTPs are polymerized onto the sequencing strand by DNA polymerase, and unbound cold dNTPs are washed away. A fluorescently labeled CoolMPS antibody mutant is then introduced, enabling non-destructive base recognition through the specific binding of cold dNTPs to the fluorescently labeled antibody. After regeneration, the antibody falls off the sequencing strand, and the newly added bases are entirely natural, without any modification. This cycle is repeated 20 times. The affinity and specificity of the CoolMPS antibody are assessed by the presence and intensity of the fluorescent signal. For specific methods, refer to the CoolMPS sequencing reagent kit instructions from BGI Genomics.

[0181] The experiment was conducted using segmented sequencing: For the first 20 sequencing cycles, four antibodies were used, all from the original kit. Subsequently, every 20 sequencing cycles, the CoolMPS antibodies targeting the blocking cytosine deoxynucleotides were the respective fluorescently labeled antibodies prepared in step one (the CoolMPS antibodies targeting the other three blocking deoxynucleotides were from the original kit). For the final 20 cycles, all four antibodies were from the original kit to correct the sequencing results and ensure no abnormalities occurred during the sequencing process. Kit information: CoolMPS High-Throughput Sequencing Kit (MGISEQ-2000RS FCL PE100), manufactured by BGI Genomics, product catalog number 1000018238.

[0182] The results showed that the sequencing fluorescence signal value RHO of the antibody mutant provided by the present invention was higher than that of Yangshen C (i.e., the original CoolMPS antibody in the kit that targets cytosine deoxynucleotides with blocking modification). Moreover, as the sequencing read length increased, the signal loss value of the antibody mutant provided by the present invention was lower than that of Yangshen C, which is beneficial for the subsequent development of long read length testing technology.

[0183] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0184] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application without departing from the principles and spirit of this application.

[0185] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An antibody that binds to a target, characterized in that: The antibody is obtained by mutating the variable region of the heavy chain of the antibody to be modified; The variable region of the heavy chain of the antibody to be modified includes three complementary determinant regions and four frame regions; the three complementary determinant regions are H-CDR1, H-CDR2, and H-CDR3; the four frame regions are H-FR1, H-FR2, H-FR3, and H-FR4; the amino acid sequence of H-CDR1 is SEQ ID NO: 1 or has more than 80% identity with SEQ ID NO: 1, the amino acid sequence of H-CDR2 is SEQ ID NO: 2 or has more than 80% identity with SEQ ID NO: 2, the amino acid sequence of H-FR1 is SEQ ID NO: 4 or has more than 80% identity with SEQ ID NO: 4, and the amino acid sequence of H-FR3 is SEQ ID NO: 6 or has more than 80% identity with SEQ ID NO: 6; The mutations are located in the following (a1) and / or (a2) and / or (a3) ​​and / or (a4) and / or (a5) and / or (a6) and / or (a7): (a1) The second amino acid residue of H-CDR1; (a2) The second amino acid residue of H-CDR2; (a3) The 15th amino acid residue of H-FR1; (a4) The 6th amino acid residue of H-FR3; (a5) The 21st amino acid residue of H-FR3; (a6) The 30th amino acid residue of H-FR3; (a7) The 38th amino acid residue of H-FR3; The target is a cytosine deoxynucleotide with blocking modification.

2. The antibody as described in claim 1, characterized in that: The mutations are as follows: (b1) and / or (b2) and / or (b3) and / or (b4) and / or (b5) and / or (b6) and / or (b7): (b1) The second amino acid residue of H-CDR1 is mutated from F to Y; (b2) The second amino acid residue of H-CDR2 is mutated from I to Y or S; (b3) The 15th amino acid residue of H-FR1 is mutated from E to G; (b4) The 6th amino acid residue W of H-FR3 is mutated to P, F, or H; (b5) The 21st amino acid residue of H-FR3 is mutated from M to T; (b6) The 30th amino acid residue of H-FR3 is mutated from D to S; (b7) The 38th amino acid residue of H-FR3 is mutated from S to Y.

3. The antigen-binding fragment of the antibody according to claim 1 or 2.

4. Antibody-related biological materials or antigen-binding fragment-related biological materials; The antibody-related biomaterials are (c1), (c2), (c3), (c4), or (c5) as follows: (c1) A polynucleotide encoding the antibody of claim 1 or 2; (c2) An expression vector having the polynucleotide described in (c1); (c3) A recombinant cell expressing the antibody of claim 1 or 2; (c4) A recombinant cell having the polynucleotide described in (c1); (c5) A recombinant cell having the expression vector described in (c2); The antigen-binding fragment-related biological material is as follows (d1), (d2), (d3), (d4), or (d5): (d1) A polynucleotide encoding the antigen-binding fragment of claim 3; (d2) An expression vector having the polynucleotide described in (d1); (d3) A recombinant cell expressing the antigen-binding fragment of claim 3; (d4) A recombinant cell having the polynucleotide described in (d1); (d5) A recombinant cell having the expression vector described in (d2).

5. A complex comprising a conjugate of substance A and substance B by chemical bonding; wherein substance A is the antibody of claim 1, the antibody of claim 2, or the antigen-binding fragment of claim 3; and substance B is a small molecule compound, a large molecule compound, or a polymer.

6. The application of antibodies or antigen-binding fragments is as follows (e1) or (e2) or (e3) or (e4): (e1) Specific binding to the target; (e2) Prepare reagents or kits for the specific binding of targets; (e3) Nucleic acid sequencing; (e4) Prepare reagents or kits for nucleic acid sequencing; The target is: cytosine deoxynucleotide with blocking modification; The antibody is the antibody of claim 1 or 2; the antigen-binding fragment is the antigen-binding fragment of claim 3.

7. A method for detecting a target, comprising the following steps: detecting whether the analyte contains a target by contacting the analyte with a label; wherein the target is a cytosine deoxynucleotide with blocking modification; wherein the label is obtained by labeling an antibody or an antigen-binding fragment; wherein the antibody is the antibody of claim 1 or 2; and wherein the antigen-binding fragment is the antigen-binding fragment of claim 3.

8. A sequencing method, comprising the following steps: The nucleic acid to be tested, four cold dNTPs and DNA polymerase were mixed and then amplified under conditions suitable for nucleic acid amplification. The amplified product after the amplification process was combined with four markers and subjected to fluorescence signal detection processing. Based on the fluorescence signal obtained from the fluorescence signal detection and processing, the nucleic acid sequence of the nucleic acid to be tested is determined; The four markers are: marker 1, obtained by labeling an antibody or antigen-binding fragment that binds to a cytosine deoxynucleotide with a blocking modification; marker 2, obtained by labeling an antibody or antigen-binding fragment that binds to an adenine deoxynucleotide with a blocking modification; marker 3, obtained by labeling an antibody or antigen-binding fragment that binds to a guanine deoxynucleotide with a blocking modification; and marker 4, obtained by labeling an antibody or antigen-binding fragment that binds to a thymine deoxynucleotide with a blocking modification. The antibody that binds to the cytosine deoxynucleotide with blocking modification is the antibody of claim 1 or 2; the antigen-binding fragment that binds to the cytosine deoxynucleotide with blocking modification is the antigen-binding fragment of claim 3.

9. A sequencing kit, characterized in that, It includes the antibody of claim 1, the antibody of claim 2, the antigen-binding fragment of claim 3, the biomaterial of claim 4, or the complex of claim 5.

10. A method for nucleotide localization, characterized in that, Includes the following steps: The primers, the nucleic acid sample to be tested, and the complex described in claim 5 are mixed together. The product of the above-mentioned mixing process is subjected to fluorescence signal detection processing; Based on the fluorescence signal obtained from the fluorescence signal detection and processing, the location of the target in the nucleic acid sample to be tested is determined; The target is a cytosine deoxynucleotide with blocking modification.

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