A method for screening molecules for binding to a target protein.

Abstract

The present invention relates to a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof, a method for screening molecules for binding to a target protein (POI) or fragment thereof using such fusion protein, and a method for screening a DNA encoding library of molecules for binding to a target protein (POI) or fragment thereof.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof, a method for screening molecules for binding to a target protein (POI) or fragment thereof using such fusion protein, and a method for screening a DNA encoding library of molecules for binding to a target protein (POI) or fragment thereof. [Background technology]

[0002] Identifying molecules that bind to target proteins is a challenging task. Techniques that facilitate the isolation of binding molecules have profound implications for pharmaceutical research, as most drug discovery programs rely on the ability to isolate small organic compounds that bind to a given protein. With the aging population and increasing understanding of disease mechanisms at the molecular level, biomedical scientists face a growing demand for more effective drugs. In addition, elucidating the biological function of proteins often requires access to specific ligands (a technique often referred to as "chemogenetics" (Strausberg, RL and Schreiber, SL, Science)). 300 (2003), 294-295). There is currently a lack of techniques for the general rapid and low-cost isolation of small organically bound molecules. Screening compound libraries for binding or inhibitory activity against target proteins is an essential part of early drug discovery. However, high-throughput screening (HTS) facilities entail a large infrastructure burden. DNA-coding libraries (DELs) offer a fundamentally different approach to early hit identification. DEL techniques utilize DNA tags to track the synthesis history of individual members in a split-and-pool combinatorial synthesis scheme. The identity of each compound is linked to its covalently bound DNA. Since the compounds are encoded in the beads, they can be pooled, and then selected for the target protein in affinity selection. After selection, the retained molecules are washed from the beads, and their DNA tags are sequenced by next-generation sequencing (NGS). DNA sequencing is the final data reading, and the hope is that the NGS read count will give a somewhat faithful representation of the binding affinity. Unfortunately, while this elegant concept works well for strong binders, information on weak binders is often below the detection threshold. Therefore, there is a need to provide effective tools and methods that enable the identification of binders, especially weak binders for the protein of interest. [Overview of the project] [Means for solving the problem]

[0003] The present invention relates to a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof, a method for screening molecules for binding to a target protein (POI) or fragment thereof using such fusion protein, and a method for screening a DNA encoding library of molecules for binding to a target protein (POI) or fragment thereof.

[0004] The inventors of this invention have developed a novel method for DEL selection that uses a DNA polymerase with terminal transferase activity to directly record binding information about DEL DNA. With this novel method, the inventors have found that, for example, equilibrium binding information leads to a more comprehensive event of the binding event, which typically differs for any target, and experimental conditions and kinetic issues (e.g., slow k under washing conditions) can be addressed. off Many of the challenges of classical selection methods for DEL, such as the problem of the affinity selection protocol itself being highly sensitive to the selection of (), have been overcome. The present invention is dedicated to the concept and proven principle for known targets. 5 This invention provides member DEL. Surprisingly, the present invention has been found to be significantly superior to state-of-the-art methods when recovering micromolar binders. Using the method of the present invention, it was found that nearly 90% of the binder was contained in the library, compared to only 38% with state-of-the-art methods.

[0005] In a first aspect, the present invention relates to a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof.

[0006] In a further embodiment, the present invention relates to a method for screening molecules for binding to a target protein (POI) or a fragment thereof, a) i) prepare an incubation medium containing deoxynucleotide triphosphates (dNTPs), ii) a fusion protein containing a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof, and iii) a conjugate compound containing the molecule and nucleic acid portion; and the steps of incubating the fusion protein and conjugate compound in the incubation medium; b) Separating the conjugate compound prepared in step a) from the conjugate compound prepared in step a) in which the nucleic acid portion is extended; c) The step of amplifying and analyzing the extended nucleic acid portion of the conjugate compound obtained in step b), which is the extended nucleic acid portion of the conjugate compound prepared in step a); d) A step of correlating the extended nucleic acid portion analyzed in step c) with the corresponding molecule of the conjugate compound used in step a), The method includes a molecule that is correlated in step d) and is selected as a POI binder.

[0007] In a further embodiment, the present invention relates to a method for screening a molecular DNA-coding library for binding to a protein of interest (POI) or a fragment thereof, a)i) an incubation medium containing deoxynucleotide triphosphates (dNTPs), ii) a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof, and iii) a DNA-coding library of chemical molecules comprising multiple entities of a single molecule in the library, each entity being covalently bonded to a nucleic acid moiety to form a conjugate compound comprising the molecule and the nucleic acid moiety; a step of incubating the fusion protein and the conjugate compound; b) separating the conjugate compound in which the nucleic acid part of the conjugate compound prepared in step a) is extended from the conjugate compound in which the nucleic acid part of the conjugate compound prepared in step a) is not extended; c) amplifying and analyzing the extended nucleic acid part of the conjugate compound obtained in step b), in which the nucleic acid part of the conjugate compound prepared in step a) is extended; d) correlating the extended nucleic acid part analyzed in step c) with a plurality of corresponding entities of a single chemical molecule of the library used in step a); The method includes the above steps, and a plurality of entities of a single chemical molecule of the library thus correlated in step d) are selected as binders for the POI.

Brief Description of the Drawings

[0008] [Figure 1] (A) Chemical structures of Conj_ID_No_4 (AAZ), Conj_ID_No_5 (CTZ), and Conj_ID_No_6 (SAA) are shown. (B) Proximity-induced extension of the DNA strand of the small molecule DNA conjugate indicates that the average extension length depends on the affinity of the small molecule fusion protein for the CAII part. [Figure 2] Concentrations of small molecule DNA conjugates Conj_ID_No_4 (AAZ), Conj_ID_No_5 (CTZ), and Conj_ID_No_6 (SAA) with respect to the concentration of unbound Conj_ID_NO_8 (amine). [Figure 3]Scatter plot of library members significantly enriched in the affinity enrichment for CAII on Ni-NTA beads. The position on the x-axis is determined by the number of compounds within diversity point 2 of the library; the position on the y-axis is determined by the number of compounds within diversity point 1 of the library. The points are adjusted according to their log-fold enrichment values. (A) Single-stranded DNA-encoded library. (B) Double-stranded DNA-encoded library. [Figure 4] Scatter plot of library members significantly enriched in the DELSTAR enrichment for CAII-SUMO-TdT of the single-stranded DNA-encoded library. The position on the x-axis is determined by the number of compounds within diversity point 2 of the library; the position on the y-axis is determined by the number of compounds within diversity point 1 of the library. The points are adjusted according to their log-fold enrichment values. (A) Standard conditions (B) 0.5 pmol of CAII-SUMO-TdT instead of 1.0 pmol. [Figure 5] Scatter plot of library members significantly enriched in the DELSTAR enrichment for CAII-SUMO-TdT of the double-stranded DNA-encoded library. The position on the x-axis is determined by the number of compounds within diversity point 2 of the library; the position on the y-axis is determined by the number of compounds within diversity point 1 of the library. The points are adjusted according to their log-fold enrichment values. (A) Standard conditions (B) 0.5 pmol of CAII-SUMO-TdT instead of 1.0 pmol. [Figure 6](A) Activity test of calmodulin-TdT compared with SUMO-TdT. The ladder (AcuteBand, LubioScience), a 50mer DNA fragment (SEQ ID NO: 13) before incubation, a 50nM concentration of this DNA with SUMO-TdT (SEQ ID NO: 7), incubation at 37°C for 30 minutes, and a 50mer DNA incubated with calmodulin-TdT (SEQ ID NO: 29) under the same conditions are shown. Extension with SUMO-TdT is slightly stronger, extending the strand by at least 50 nucleotides. Calmodulin extends the strand by at least 40 nucleotides. (B) Incubation of calmodulin-TdT with DBCO-DNA or DNA conjugated with calmodulin-binding peptide under standard conditions. The lanes indicate that proximity-induced extension is stronger for DNA-calmodulin-binding peptide conjugates (SEQ ID NO: 33 and SEQ ID NO: 13) compared to background extension for DBCO-DNA (SEQ ID NO: 13). (C) SDS-PAGE shows SpyTag-CAII (lane 2), SpyCatcher-SUMO-TdT (lane 3), and their conjugate fusions (lane 4). Conversion to complete conjugates is not complete. (D) Proximity-induced extension assay under standard conditions using SpyTag-CAII-SpyCatcher-TdT shows the formation of proximity-induced products of CAII-binding small molecule conjugates AAZ (conjugate 1), CTZ (conjugate 2), and SAA (3, see Table VII). Unbound DBCO-DNA (SEQ ID NO: 16) does not show extension after pull-down with dT25 beads. [Figure 7](A) A model system that emulates POI-small molecule interactions by using DNA-DNA base pairing. DNA-SNAP-TdT adducts are incubated with DNA fragments adjusted to a specific dissociation constant simulated by introducing mismatches within the sequence. (B) As the dissociation constant increases, the tail attached to the DNA becomes smaller. When SNAP-TdT is used directly instead of DNA-SNAP-TdT, non-proximity-induced extension is minimal, slightly less than in the case of the weakest interaction. [Modes for carrying out the invention]

[0009] As outlined above, the present invention provides a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof, a method for screening molecules for binding to a target protein (POI) or fragment thereof using such fusion protein, and a method for screening a DNA encoding library of molecules for binding to a target protein (POI) or fragment thereof.

[0010] Accordingly, in a first aspect, the present invention provides a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof.

[0011] For the purposes of interpreting this specification, the following definitions apply, and where appropriate, a singular term also includes a plural form, and vice versa. It should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0012] Features, integers, properties, and compounds described in conjunction with specific aspects, embodiments, or examples of the present invention should be understood to be applicable to any other aspects, embodiments, or examples described herein, insofar as they are not incompatible therewith. All features disclosed herein (including any appended claims, abstract, and drawings) and / or all steps of any methods or processes so so may be combined in any combination except in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of any of the embodiments described herein.

[0013] The terms "comprise" and its variations, such as "comprises" and "comprising," are generally used to mean "to include, but not limited to," that is, to allow the presence of one or more features or components.

[0014] The singular forms "a," "an," and "the" refer to multiple objects unless otherwise specified by the context.

[0015] The term "approximately" refers to a range of ±10% of a specific value. For example, the phrase "approximately 200" includes ±10% of 200, i.e., 180 to 220.

[0016] The term “DNA-coding library of chemical molecules” or the abbreviation “DEL,” as used herein, refers to a library of chemical molecules covalently bound to nucleic acid portions, such as short nucleic acids, which serve as identification barcodes and, optionally, further direct and control chemosynthesis. The technology typically enables mass creation via split-and-pool synthesis and library matching via affinity selection against immobilized protein targets. In the present invention, double-stranded (ds) DNA-coding libraries of chemical molecules and single-stranded (ss) DNA-coding libraries of chemical molecules may be used, and preferably, single-stranded (ss) DNA-coding libraries of chemical molecules are used.

[0017] The term “DNA polymerase or fragment thereof having terminal transferase activity” means, as used herein, a DNA polymerase or fragment thereof that catalyzes the addition of a deoxynucleotide to the 3' hydroxyl end of a DNA molecule. Preferred DNA polymerases or fragments thereof having terminal transferase activity are terminal deoxynucleotidyltransferases (TDTs) or fragments thereof. Fragments of DNA polymerases having terminal transferase activity typically contain 100 to 1000 amino acids, preferably 200 to 600 amino acids, more preferably 300 to 500 amino acids, even more preferably 350 to 400 amino acids, and even more preferably about 379 amino acids.

[0018] The term “terminal deoxynucleotidyltransferase (TDT) or fragment thereof,” also known as DNA nucleotidyl exotransferase (DNTT) or terminal transferase, as used herein, refers to a DNA polymerase that catalyzes the addition of nucleotides to the 3' end of a DNA molecule. Unlike most DNA polymerases, it does not require a template. The preferred substrate for this enzyme is the 3'-overhang, but it can also add nucleotides to a blunt or recessed 3' end. Fragments of TDT typically contain 100 to 1000 amino acids, preferably 200 to 600 amino acids, more preferably 300 to 500 amino acids, even more preferably 350 to 400 amino acids, and even more preferably about 379 amino acids.

[0019] The term “Protein of Interest (POI) or its fragment” as used herein refers to any protein, preferably a human pharmacokinetic protein, i.e., a therapeutic protein or therapeutic enzyme. Examples of human pharmacokinetic proteins include cytokines, growth factors, hormone antibodies, or vaccines. The terms “its fragment” and “functional fragment” are used equally herein. The term “Protein of Interest (POI) or its fragment” includes naturally occurring POIs as well as artificially modified POIs. Artificially, a POI is, for example, a variant or functional fragment of a POI. In relation to the POI of this invention, “its variant or functional fragment” means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the POI. Such variants include naturally occurring allelic variants and variants not naturally occurring. Addition, deletion, substitution, and derivatization of one or more amino acids are intended, provided that the modification does not result in a deficiency of the functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity to the relevant portion of the POI, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98%. The POI fragments as defined herein certainly have the same functional properties as the POI from which they are derived. POIs typically contain 100 to 1000 amino acids, preferably 200 to 800 amino acids, more preferably 300 to 700 amino acids, and even more preferably 300 to 600 amino acids. The POI fragments typically contain 25 to 500 amino acids, preferably 50 to 400 amino acids, more preferably 100 to 300 amino acids, and even more preferably 100 to 200 amino acids.

[0020] The term "linker between a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof" as used herein refers to a linker, for example, a linker or a linker comprising 1 to 200, preferably 5 to 150, more preferably 10 to 150 amino acids or a chemical entity. The linker is typically introduced between the amino terminus of a DNA polymerase having terminal transferase activity and the carboxyl terminus of a protein of interest (POI).

[0021] The term "conjugate compound comprising a binder for POI or a fragment thereof and a nucleic acid moiety" as used herein refers to a compound in which the binder for POI or a fragment thereof is coupled to the nucleic acid moiety, preferably covalently, by coupling DBCO-modified DNA with an azide-functionalized binder for POI or a fragment thereof.

[0022] The term "POI binder," as used herein, refers to a molecule capable of binding to a POI, such as a chemical entity or peptide, preferably a molecule capable of binding to a POI such that the binding causes modification of the POI's activity and / or chemical structure, such as a chemical entity or peptide. A preferred POI binder is a chemical entity.

[0023] The term “nucleic acid moiety,” as used herein, refers to a unit or component at the nucleic acid level, including coding and non-coding nucleic acid moieties. Preferably, the nucleic acid moiety is DNA, more preferably ssDNA or dsDNA, and even more preferably ssDNA. More preferably, the nucleic acid moiety is a non-coding nucleic acid moiety, and even more preferably, the nucleic acid moiety is non-coding DNA, particularly non-coding ssDNA or dsDNA, and more specifically non-coding ssDNA. The nucleic acid moiety of a conjugate compound typically contains 5 to 1500, 10 to 100, 10 to 500, or 5 to 200 nucleic acid bases, preferably 5 to 1500 nucleic acid bases. When the nucleic acid moiety of a conjugate compound interacts with the DNA polymerase of the fusion protein, it typically results in poly-A tailing of the POI or its fragment binder.

[0024] The term "tag," as used herein, may encompass affinity tags, solubilization tags, chromatography tags, and epitope tags. Adding / fusioning affinity tags (also used as purification tags) to proteins enables the purification of tagged molecules from their unpurified biosources using affinity purification techniques. These include glutathione-S-transferase (GST), biotin, modified biotin, and poly(His) tags. Poly(His) tags are widely used; they bind to metal-containing substrates. Solubilization tags are used to assist recombinant proteins, particularly those expressed in chaperone-deficient species such as Escherichia coli (E. coli), in proper protein folding and prevent precipitation. These include thioredoxin (TRX) and poly(NANP).

[0025] In some embodiments, the fusion protein comprises a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof, provided that the fusion protein is not a fusion protein containing terminal deoxynucleotidyltransferase (TDT) and Cas9.

[0026] In some embodiments, the fusion protein comprises a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof, provided that the fusion protein is not a fusion protein containing a terminal deoxynucleotidyltransferase (TDT) and a SUMO protein or fragment thereof.

[0027] In some embodiments, the fusion protein comprises a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof, provided that the fusion protein is not a fusion protein comprising a terminal deoxynucleotidyltransferase (TDT) and a SUMO protein or fragment thereof, and provided that the fusion protein is not a fusion protein comprising a terminal deoxynucleotidyltransferase (TDT) and a glutathione S-transferase or fragment thereof.

[0028] In some embodiments, the fusion protein comprises a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof, provided that the fusion protein is not a fusion protein comprising terminal deoxynucleotidyltransferase (TDT) and Cas9, provided that the fusion protein is not a fusion protein comprising terminal deoxynucleotidyltransferase (TDT) and SUMO protein or a fragment thereof, and provided that the fusion protein is not a fusion protein comprising terminal deoxynucleotidyltransferase (TDT) and glutathione S-transferase or a fragment thereof.

[0029] In some embodiments, a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof comprises a DNA polymerase fragment having terminal transferase activity and a protein of interest (POI) or fragment thereof, preferably a fragment of terminal deoxynucleotidyltransferase (TDT) and a protein of interest (POI) or fragment thereof. The DNA polymerase fragment having terminal transferase activity typically comprises a C-terminal POLXc domain and, more specifically, an 8kDa- domain, a finger domain, a palm domain, and a subdomain. The DNA polymerase fragment having terminal transferase activity is preferably a fragment of terminal deoxynucleotidyltransferase (TDT) having 43kDa.

[0030] In some embodiments, a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof comprises a DNA polymerase or fragment thereof having terminal transferase activity, preferably a fragment of DNA polymerase having terminal transferase activity, and two or more copies of the protein of interest (POI) or two or more copies of fragment thereof, preferably a terminal deoxynucleotidyltransferase (TDT) or fragment thereof, preferably a fragment of terminal deoxynucleotidyltransferase (TDT) and two or more copies of the protein of interest (POI) or two or more copies of fragment thereof.

[0031] In some embodiments, the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and the target protein (POI) or fragment thereof comprises two or more copies of the DNA polymerase having terminal transferase activity or two or more copies of a fragment of the DNA polymerase having terminal transferase activity, preferably a fragment of the DNA polymerase having terminal transferase activity and two or more copies of the target protein (POI) or fragment thereof, preferably two or more copies of the terminal deoxynucleotidyltransferase (TDT) or two or more copies of a fragment thereof, preferably a fragment of the terminal deoxynucleotidyltransferase (TDT) and two or more copies of the target protein (POI) or fragment thereof.

[0032] In some embodiments, the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof comprises two or more copies of the DNA polymerase having terminal transferase activity or two or more copies of a fragment of the DNA polymerase having terminal transferase activity, preferably two or more copies of a fragment of the DNA polymerase having terminal transferase activity and two or more copies of a protein of interest (POI) or fragment thereof, preferably two or more copies of a terminal deoxynucleotidyltransferase (TDT) or two or more copies of a fragment thereof, preferably two or more copies of a fragment of a terminal deoxynucleotidyltransferase (TDT) and two or more copies of a protein of interest (POI) or two or more copies of a fragment thereof.

[0033] Two or more copies of the target protein (POI) or two or more copies of its fragments are preferably two copies of the target protein (POI) or two copies of its fragments, three copies of the target protein (POI) or three copies of its fragments, four copies of the target protein (POI) or four copies of its fragments, or five copies of the target protein (POI) or five copies of its fragments, more preferably two to five copies of the target protein (POI) or two to five copies of its fragments. A copy of the target protein or a copy of its fragments usually contains multiple identical protein sequences of the target protein or multiple identical protein sequences of its fragments.

[0034] Two or more copies of DNA polymerase having terminal transferase activity or two or more copies of DNA polymerase fragments having terminal transferase activity are preferably two copies of DNA polymerase having terminal transferase activity or two copies of DNA polymerase fragments having terminal transferase activity, three copies of DNA polymerase having terminal transferase activity or three copies of DNA polymerase fragments having terminal transferase activity, four copies of DNA polymerase having terminal transferase activity or four copies of DNA polymerase fragments having terminal transferase activity, five copies of DNA polymerase having terminal transferase activity or five copies of DNA polymerase fragments having terminal transferase activity, and more preferably two to five copies of DNA polymerase having terminal transferase activity or two to five copies of DNA polymerase fragments having terminal transferase activity. A copy of a DNA polymerase having terminal transferase activity, or a copy of a fragment thereof, typically contains multiple identical nucleic acid sequences of the DNA polymerase having terminal transferase activity, or multiple identical nucleic acid sequences of the fragment thereof.

[0035] In some embodiments, the fusion protein includes a linker between a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof. Preferably, the linker includes 1 to 300 amino acids, preferably 10 to 150 amino acids, more preferably 30 to 150 amino acids, and even more preferably 40 to 150 amino acids, or the linker includes or is a chemical entity. In one embodiment, the linker includes a SUMO protein or fragment thereof, e.g., a SUMO tag, and includes 1 to 300 amino acids, preferably 10 to 150 amino acids, and more preferably the amino acid sequence shown in SEQ ID NO: 6. In a further embodiment, the linker includes SEQ ID NO: 44. In a further embodiment, the linker includes a SpyTag connected to a SpyCatcher[8], and the linker preferably includes an amino acid sequence shown in SEQ ID NO: 45 connected to an amino acid sequence shown in SEQ ID NO: 46, and preferably the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 46 are linked via an isopeptide bond between the first D from the amino terminus of SEQ ID NO: 45 and the second K from the amino terminus of SEQ ID NO: 46. In preferred embodiments, the linker is selected from the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 45, and combinations thereof. In some embodiments, the linker comprises a chemical entity. Thus, in some embodiments, the fusion protein comprises a linker between a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof, where the linker is a chemical entity. Chemical entities usable as linkers in the present invention are usually common bioconjugation motifs known to those skilled in the art, and are preferably selected from the group consisting of maleimide-benzylguanine, maleimide-hexylchloride, activated ester-benzylguanine, and activated ester-hexylchloride conjugates, and preferentially have a short carbon or PEG linker.

[0036] In a preferred embodiment, the fusion protein comprises one or more, preferably two to five, linkers between a DNA polymerase or fragment thereof having terminal transferase activity and the target protein (POI) or fragment thereof, wherein the linkers are chemical entities, and one or more, preferably two to five, linkers are fused in a portion of the linker to amino acids of the target protein (POI) or fragment that are different from the N-terminal and / or C-terminal amino acids of the target protein (POI) or fragment thereof, and the same one or more linkers are fused in another portion of the linker to a DNA polymerase or fragment thereof having terminal transferase activity.

[0037] In some embodiments, a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof includes a tag. The tag can be located at the N-terminus or C-terminus of the fusion protein, preferably at the N-terminus.

[0038] In some embodiments, the DNA polymerase or fragment thereof having terminal transferase activity is a terminal deoxynucleotidyltransferase (TDT) or fragment thereof, preferably a terminal deoxynucleotidyltransferase (TDT) or fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 5.

[0039] In some embodiments, the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof comprises a DNA polymerase or fragment thereof having terminal transferase activity, preferably a terminal deoxynucleotidyltransferase (TDT) or fragment thereof, preferably a terminal deoxynucleotidyltransferase (TDT) or fragment thereof included by the amino acid sequence shown in SEQ ID NO: 5, and a calmodulin or fragment thereof, preferably a calmodulin or fragment thereof included by the amino acid sequence shown in SEQ ID NO: 43. In preferred embodiments, the fusion protein comprises a DNA polymerase or fragment thereof having terminal transferase activity and a calmodulin or fragment thereof. In particular, the fusion protein comprises a terminal deoxynucleotidyltransferase (TDT) or fragment thereof and a calmodulin or fragment thereof shown in SEQ ID NO: 29.

[0040] In some embodiments, the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof comprises a sequence selected from the group shown by SEQ ID NOs: 7 to 12, preferably a sequence selected from the group shown by SEQ ID NOs: 8, 9, 11 and 12, particularly SEQ ID NOs: 11 or 12, more preferably a sequence selected from the group shown by SEQ ID NOs: 8, 9, 11, 12, 29, 31 and 32, particularly SEQ ID NOs: 11, 12, 29 or 31, and even more preferably a sequence selected from the group shown by SEQ ID NOs: 8, 9, 11, 12 and 29, particularly SEQ ID NOs: 11, 12 or 29.

[0041] In some embodiments, the POI or fragment thereof is selected from the group consisting of enzymes or fragments thereof and proteins or fragments thereof that target or are involved in cell proliferation, preferably proteins or fragments thereof that target or are involved in cell proliferation. More preferably, the POI or fragment thereof is a protein or fragment thereof that targets or is involved in cell proliferation, selected from the group consisting of IL2, RIPK1, MAPK14, PARP1, SIRT3, PI3Kbeta, PI3Kgamma, PI3Kdelta, BRCA1, BRCA2, BRAT1, INTS9, and INTS11, fragments thereof, or variants thereof. Even more preferably, the POI is selected from the group consisting of carbonic anhydrase II (CAII), Rad52, Rad51, KRAS-G12C, PI3Kalpha, PALB2, and DCAF15, fragments thereof, or variants thereof.

[0042] In a further embodiment, the present invention provides a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof, which is conjugated to a conjugate compound comprising a binder for the POI or fragment and a nucleic acid moiety. "The fusion protein is conjugated to a conjugate compound comprising a binder for the POI or fragment and a nucleic acid moiety" means, as used herein, that the fusion protein and the conjugate compound may be linked by non-covalent or covalent bonds. Non-covalent bonds include pp (aromatic) interactions, van der Waals interactions, H-bond interactions, and ionic interactions. Preferably, the fusion protein and the conjugate compound are linked by non-covalent bonds. The fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof is as described above.

[0043] In some embodiments, the conjugate compound comprising a binder and a nucleic acid moiety of a POI or fragment thereof is a conjugate in which the binder is covalently coupled to the nucleic acid moiety. In some embodiments, the binder of the POI or fragment thereof is a chemical entity or a peptide, preferably a chemical entity. In some embodiments, the binder of the POI or fragment thereof is a peptide selected from the group consisting of streptag, streptavidin, streptactin, SnoopTag, SnoopCachter, and a calmodulin-binding peptide. Preferably, the peptide is a calmodulin-binding peptide, more preferably the calmodulin-binding peptide shown in SEQ ID NO: 33. In some embodiments, the nucleic acid moiety of the conjugate is ssDNA or dsDNA, preferably ssDNA. In some embodiments, the conjugate comprises DNA and a chemical entity shown in SEQ ID NOs: 13-15 or SEQ ID NOs: 16-18.

[0044] Accordingly, in a further embodiment, the present invention provides a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein or fragment thereof, the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein or fragment thereof, which is bound to a further fusion protein comprising a peptide and a POI or fragment thereof, where the peptide (petide) has the ability to bind to the protein or fragment thereof of the fusion protein. The protein or fragment thereof is typically selected from proteins or fragments thereof that have a corresponding binding peptide, such as a protein or fragment thereof that is part of a known binding system, such as streptavidin or streptactin, SnoopTag or SnoopCachter, or calmodulin or calmodulin-binding peptide, and which has the ability to bind to the protein or fragment thereof of the fusion protein that is the other part of these binding systems. In one embodiment, the fusion protein comprises a DNA polymerase or fragment thereof having terminal transferase activity and calmodulin or fragment thereof, and the fusion protein comprising the DNA polymerase or fragment thereof having terminal transferase activity and calmodulin or fragment thereof is bound to a further fusion protein comprising a calmodulin-binding peptide and POI or fragment thereof. "The fusion protein comprising the DNA polymerase or fragment thereof having terminal transferase activity and calmodulin or fragment thereof is bound to a further fusion protein comprising a calmodulin-binding peptide and POI or fragment thereof" means, as used herein, that the (first) fusion protein and the further (second) fusion protein may be linked by non-covalent or covalent bonds. Non-covalent bonds include pp (aromatic) interactions, van der Waals interactions, H-bond interactions, and ionic interactions. Preferably, the (first) fusion protein and the further (second) fusion protein are linked by non-covalent bonds. The DNA polymerase or fragment thereof having terminal transferase activity, the target protein (POI) or fragment thereof, calmodulin or fragment thereof, and calmodulin-binding peptide are as described above.

[0045] In a further embodiment, the present invention relates to a method for screening molecules for binding to a target protein (POI) or a fragment thereof, a) i) prepare an incubation medium containing deoxynucleotide triphosphates (dNTPs), ii) a fusion protein containing a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof, and iii) a conjugate compound containing the molecule and nucleic acid portion; and the steps of incubating the fusion protein and conjugate compound in the incubation medium; b) Separating the conjugate compound prepared in step a) from the conjugate compound in which the nucleic acid portion is extended from the conjugate compound in which the nucleic acid portion is not extended; c) The step of amplifying and analyzing the extended nucleic acid portion of the conjugate compound obtained in step b), which is the extended nucleic acid portion of the conjugate compound prepared in step a); d) A step of correlating the extended nucleic acid portion analyzed in step c) with the corresponding molecule of the conjugate compound used in step a), The method provides a method in which the molecule correlated in step d) is selected as a POI binder.

[0046] The DNA polymerase or fragment thereof having terminal transferase activity and the fusion protein comprising the target protein (POI) or fragment thereof, as well as the conjugate compound comprising the molecule and nucleic acid portion to be used for the method, are as described above.

[0047] The incubation typically includes dATP, dCTP, dGTP, and / or dTTP, and preferably the incubation medium contains dATP. The fusion protein and conjugate compound are typically incubated in the incubation medium for 10 to 60 minutes, preferably at 37°C for 30 minutes.

[0048] In some embodiments of step a), the DNA polymerase or fragment thereof having terminal transferase activity is inactivated before step b), preferably by heat treatment, more preferably by heating the incubation of step a) at 98°C for 10 minutes before step b).

[0049] In some embodiments, step b) includes adding a solid phase containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound to an incubation medium, and separating the solid phase containing the deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound that has been hybridized to the extended nucleic acid portion of the conjugate compound from the solid phase containing the deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound that has not been hybridized to the extended nucleic acid portion of the conjugate compound.

[0050] In some embodiments, step b) includes adding polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound to an incubation medium, and separating the polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound that have been hybridized to the extended nucleic acid portion of the conjugate compound from the polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound that have not been hybridized to the extended nucleic acid portion of the conjugate compound.

[0051] In some embodiments, the conjugate compound having an extended nucleic acid moiety is eluted from a solid phase containing a deoxynucleotide complementary to the extended nucleic acid moiety of the conjugate compound, or from polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid moiety of the conjugate compound.

[0052] In some embodiments, the washing step is performed after the conjugate compound having an extended nucleic acid portion has been eluted from a solid phase containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound, or from polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound.

[0053] In some embodiments of the conjugate compound in which the nucleic acid moiety of the conjugate compound prepared in step a) is extended, the nucleic acid moiety is extended by 1 to 200 nucleotides, preferably 10 to 100 nucleotides, and more preferably 20 to 50 nucleotides.

[0054] In some embodiments, the extended nucleic acid portion is amplified and sequenced in step c).

[0055] The correlation between the extended nucleic acid portion analyzed in step c) and the corresponding molecule of the conjugate compound used in step a) is usually performed by measuring the concentration of individual strands, preferably by measuring the concentration of specific strands by quantitative PCR using selective primers, or by counting the occurrence of barcodes after sequencing and comparing them with the results without the target protein.

[0056] In a further embodiment, the present invention relates to a method for screening a molecular DNA-coding library for binding to a protein of interest (POI) or a fragment thereof, a)i) an incubation medium containing deoxynucleotide triphosphates (dNTPs), ii) a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof, and iii) a DNA-coding library of chemical molecules comprising multiple entities of a single molecule in the library, each entity being covalently bonded to a nucleic acid moiety to form a conjugate compound comprising the molecule and the nucleic acid moiety; a step of incubating the fusion protein and the conjugate compound; b) Separating the conjugate compound prepared in step a) from the conjugate compound in which the nucleic acid portion is extended from the conjugate compound in which the nucleic acid portion is not extended; c) The step of amplifying and analyzing the extended nucleic acid portion of the conjugate compound obtained in step b), which is the extended nucleic acid portion of the conjugate compound prepared in step a); d) The step of correlating the extended nucleic acid portion analyzed in step c) with multiple corresponding entities of a single chemical molecule in the library used in step a), The method provides a way in which multiple entities of a single chemical molecule in the library, correlated in this way in step d), are selected as binders for the POI.

[0057] A DNA-coding library of chemical molecules may be a double-stranded (ds) DNA-coding library of chemical molecules or a single-stranded (ss) DNA-coding library of chemical molecules, but is preferably a single-stranded (ss) DNA-coding library of chemical molecules.

[0058] The incubation typically includes dATP, dACTP, dGTP, and / or dTTP, and preferably the incubation medium contains (dATP). The fusion protein and conjugate compound are typically incubated in the incubation medium for 10 to 60 minutes, preferably at 37°C for 30 minutes.

[0059] The DNA polymerase or fragment thereof having terminal transferase activity and the fusion protein comprising the target protein (POI) or fragment thereof, as well as the conjugate compound comprising the molecule and nucleic acid portion to be used for the method, are as described above.

[0060] In some embodiments of step a), the DNA polymerase or fragment thereof having terminal transferase activity is inactivated before step b), preferably by heat treatment, more preferably by heating the incubation of step a) at 98°C for 10 minutes before step b).

[0061] In some embodiments, step b) includes adding a solid phase containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound to an incubation medium, and separating the solid phase containing the deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound that has been hybridized to the extended nucleic acid portion of the conjugate compound from the solid phase containing the deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound that has not been hybridized to the extended nucleic acid portion of the conjugate compound.

[0062] In some embodiments, step b) includes adding polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound to an incubation medium, and separating the polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound that have been hybridized to the extended nucleic acid portion of the conjugate compound from the polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound that have not been hybridized to the extended nucleic acid portion of the conjugate compound.

[0063] In some embodiments, the conjugate compound having an extended nucleic acid moiety is eluted from a solid phase containing a deoxynucleotide complementary to the extended nucleic acid moiety of the conjugate compound, or from polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid moiety of the conjugate compound.

[0064] In some embodiments, the washing step is performed after the conjugate compound having an extended nucleic acid portion has been eluted from a solid phase containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound, or from polydeoxynucleotide beads containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound.

[0065] In some embodiments of the conjugate compound in which the nucleic acid moiety of the conjugate compound prepared in step a) is extended, the nucleic acid moiety is extended by 1 to 200 nucleotides, preferably 10 to 100 nucleotides, and more preferably 20 to 50 nucleotides.

[0066] In some embodiments, the extended nucleic acid portion is amplified and sequenced in step c).

[0067] The correlation between the extended nucleic acid portion analyzed in step c) and the corresponding molecule of the conjugate compound used in step a) is typically performed by measuring the concentration of individual strands, preferably by measuring the concentration of specific strands by quantitative PCR using selective primers, or by counting the occurrence of barcodes after sequencing and comparing them with results without the target protein.

[0068] The library used in the above-described method of the present invention provides a repertoire of chemical diversity such that each chemical part is linked to a DNA part that facilitates the identification of the chemical part.

[0069] This screening method makes it possible to identify optimized chemical structures involved in binding interactions with target proteins by utilizing a repertoire of structures randomly formed by the association of diverse components, eliminating the need to synthesize them one by one or to recognize their interactions beforehand.

[0070] The incubation of the library with other prepared components may result in a heterogeneous or homogeneous mixture. Therefore, the library members may be present in the solid phase while the fusion protein is present in the liquid phase. Alternatively, the fusion protein may be present in the solid phase while the library members are present in the liquid phase. Furthermore, both the library members and the fusion protein may be present in the liquid phase.

[0071] Binding conditions are those that conform to the known innate binding function of POIs. These conforming conditions are buffer, pH, and temperature conditions that maintain the biological activity of DNA polymerases with terminal transferase activity of POIs and their fragments, thereby maintaining the molecule's ability to participate in its pre-selected binding interactions. Typically, these conditions include physiological aqueous solutions with pH and ionic strength usually associated with POIs.

[0072] The use of solid supports is generally well known in the art. Useful solid support matrices are well known in the art and include crosslinked dextran, for example, available under the trade name SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ); agarose, borosilicate, polystyrene or latex beads (with a diameter of about 1 micron to about 5 millimeters), polyvinyl chloride, polystyrene, crosslinked polyacrylamide, nitrocellulose or nylon-based webs, for example, insoluble matrices such as sheets, strips, paddles, and microtiter plate wells of plates. [Examples]

[0073] Example 1: A) Materials and methods Bacterial strains and growth conditions. For plasmid purification and cloning, Escherichia coli (E. coli) DH5α was grown at 37°C on LB agar and in LB culture medium. Ampicillin was used at a concentration of 100 μg / mL to select the expression vector. For protein expression, Escherichia coli (E. coli) BL21 (DE3) was used under the same conditions, and induction was performed using 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.5-0.6.

[0074] Plasmid construction. The pET19b vector backbone was used for all expression. The sequence of the thermostable terminal dideoxynucleotide transferase used (hereafter TdTevo, SEQ ID NO: 5) was adapted from Barthel et al. [1]. The sequence of carbonic anhydrase II (hereafter CAII) was donated by Ryan Mehl (RRID: Addgene_105665). The linker used between TdT and the target protein is shown in SEQ ID NO: 6. Table I shows the nucleotide sequences inserted into the backbone for all fusion proteins, including the first nucleotide of the NcoI restriction site and the final nucleotide of the BamHI restriction site; Table II shows the corresponding plasmids and fusions; and Table III shows the corresponding protein sequences.

[0075] [Table 1]

[0076] [Table 2]

[0077] [Table 3]

[0078] [Table 4]

[0079] Table 5

[0080] Table 6

[0081] Table 7

[0082] Table 8

[0083] Table 9

[0084] Table 10

[0085] Protein Expression. Proteins were expressed in *E. coli* BL21 (DE3) containing each protein sequence summarized in Table II. For CAII plasmids (Seq_ID_2 and Seq_ID_4), bacteria were grown in 400 mL of LB medium supplemented with ampicillin (100 μg / mL) and 400 μM ZnCl2 at 37°C until the OD600 reached 0.5-0.6. The cultures were induced with IPTG (0.5 mM), and proteins were expressed at 250 rpm at 18°C ​​for 18 hours. Cells were collected and resuspended in a lysis buffer containing pH 8.0 Tris-HCl (100 mM), sucrose (100 g / L), glycerol (0.1 L / L), sodium chloride (1 M), and one tablet of a completely EDTA-free protease inhibitor cocktail (Roche). The cells were then lysed by sonication at 4°C (Hielscher UP200St, 5 cycles (10 seconds at maximum power, 150 seconds pause)). After centrifugation at maximum speed (Centrifuge 5418R, Eppendorf), the supernatant was collected, and the proteins were purified using His / Ni beads (ROTI® Garose), followed by trace elution of bound proteins. The fraction containing the desired protein was pooled, and His-CAII (Seq_ID_10) was excluded when 10,000 MWCO was used instead. The fraction was then concentrated using a centrifugal concentrator (Sartorius Vivaspin 500 or Vivaspin 2) with a suitable MW cutoff of 50,000 MWCO and a PES membrane to obtain the desired protein in a storage buffer containing 200 mM KH2PO4 and 100 mM NaCl at pH 6.5.

[0086] For His-CAII (Seq_ID_10) and His-SUMO-TdTEvo (Seq_ID_7), the proteins were divided equally and rapidly frozen at -80°C.

[0087] For His-SUMO-CAII-SUMO-TdTEvo (Seq_ID_8) and His-SUMO-SNAP-SUMO-TdTEvo (Seq_ID_9), the proteins were first incubated with TEV protease at 4°C according to the manufacturer's procedure (New England Biolabs). The reaction mixture was purified with His / Ni beads (Rotigarose), and the flow-through was concentrated instead, with the buffer replaced again with storage buffer. The proteins were then divided into equal portions, rapidly frozen, and stored at -80°C.

[0088] The protein sequences are summarized in Table IV. The cleavage products for TEV are summarized in Table V.

[0089] [Table 11]

[0090] [Table 12]

[0091] Deprotection of Acetazolamide 1 [ka] Follow the procedure according to More et al.[2] A suspension of acetazolamide (1 g, 4.00 mmol) in EtOH (20 mL) was mixed with concentrated hydrochloric acid (5 mL), and the mixture was back-flowed for 6 hours. The solvent was evaporated, saturated NaHCO3 was added, and the mixture was extracted with phenylethylamine. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to obtain sulfonamide 2 (0.35 g, 50%) as a white solid. 1 H-NMR (400MHz, DMSO-d6): δ (ppm) 8.04 (s, 2H), 7.82 (s, 2H).

[0092] Synthesis of acetazolamide carboxylic acid 4

Chemical formula

[0093] Synthesis of acetazolamide azide 6

Chemical formula

[0094] Synthesis of azido-linked chlorothiazide 9 Synthesis of compound chlorothiazide precursor 8 [ka] Qualification procedure according to Katzl and Ruis.[3] Sulfonamide 7 (2.00 g, 7.00 mmol, 1.0 equivalent) and anhydrous 3 (6.00 g, 60.2 mmol, 8.6 equivalents) were heated to 195°C over 60 minutes. After cooling the melt, it was suspended in warm H2O (55°C), and the precipitate was thoroughly filtered. It was dried on a high-temperature surface to obtain intermediate 8 (1.3 g, 53%) as a white solid. 1 H-NMR (500MHz, DMSO-d6): δ(ppm)8.97(s,1H),8.41(s,1H),8.05(s,2H),3.11-3.08(m,2H),2.91-2.89(m,2H). HRMS(ESI,m / z):C 10 H8ClN3O5S2H + :349.9594 (calculated value), 381.9753 (measured value + MeOH).

[0095] Synthesis of chlorothiazide 9 [ka] Intermediate 8 (15 mg, 42.9 μmol, 1.0 equivalent), azid linker 5 (14 mg, 12.8 μL, 64.3 μmol, 1.5 equivalents), and DIPEA (16.6 mg, 22.4 μL, 129 μmol, 3 equivalents) were dissolved in DMF (500 μL) and stirred at room temperature for 18 hours. The crude mixture was directly purified by preparative HPLC to obtain product 9 (14 mg, 58%) as a white solid. 1 H-NMR(500MHz,DMSO-d6):δ(ppm)12.43(s,1H),8.25(s,1H),8.05(t, 3 J HH =5.6Hz,1H),7.88(s,2H),7.48(s,1H),3.60-3.58(m,2H),3.57-3.48(m,8H),3.41-3.38(m,4H),3.20(q, 3 J HH =5.7Hz,2H),2.83(t, 3 J HH =7.2Hz,2H),2.55(t, 3 J HH (=7.2Hz, 2H). 13 C-NMR(126MHz,DMSO-d6):δ(ppm)170.4,160.9,138.4,138.3,134.4,125.0 ,119.9,119.0,69.8,69.8,69.7,69.6,69.2,69.1,50.0,38.7,30.8,30.4. HRMS(ESI,m / z):C 18 H 25 ClN7O8S2 - : 566.0973 (calculated value), 566.0900 (measured value).

[0096] Synthesis of azide-bonded sulfanilamide 12 Synthesis of sulfanilamide carboxylic acid 11 [ka] The procedure according to Akocak et al.[4] Phenylsulfonamide 10 (5.00 g, 29.9 mmol, 1.0 equivalent) and anhydrous 3 (3.20 g, 32.0 mmol, 1.1 equivalents) were mixed in MeCN (50 mL) and backflowed for 16 hours. The reaction mixture was allowed to cool to room temperature, and the precipitate was thoroughly filtered to obtain product 11 (7.26 g, 93%) as a white solid. 1H-NMR (400MHz, DMSO-d6): δ(ppm)12.15(s,1H),10.30(s,1H),7.73(s,4H),7.23(s,2H),2.63-2.57(m,2H),2.56-2.52(m,2H).

[0097] Synthesis of sulfanilamide azide 12 [ka] Intermediate 11 (63 mg, 232 μmol, 1.0 equivalent), azid linker 5 (45.5 mg, 50 mL, 208 μmol, 0.9 equivalent), and EDC HCl (45 mg, 232 μmol, 1.0 equivalent) were dissolved in DMF (1.0 mL) and stirred at room temperature for 18 hours. After removing the solvent, the crude product was purified by preparative HPLC to obtain product 12 (49 mg, 50%) as a white solid. 1 H-NMR(400MHz,DMSO-d6):δ(ppm)10.28(s,1H),7.93(s,1H),7.74(s,4H),7.23(s,2H),3.67-3.46(m,10H),3.44-3.31(m,4H),3.19(q, 3 J HH =5.9Hz,2H),2.58(t, 3 J HH =7.0Hz,2H),2.42(t, 3 J HH (=7.0Hz, 2H). 13 C-NMR (101MHz, DMSO-d6): δ(ppm)171.2,171.1,142.3,138.0,126.7,118.4,69.8,69.8,69.7,69.6,69.3,69.1,50.0,38.6,31.7,30.0.

[0098] Synthesis of small molecule nucleic acid conjugates. DNA with 5'DBCO modification (30 μL, 100 μM (in water), 1.0 equivalent, Microsynth) was incubated with azide-functionalized small molecules (3 μL, 10 mM (in DMSO), 10 equivalents) and MOPS buffer (2 μL, 50 mM MOPS, 500 mM NaCl, pH 8.2) at room temperature for 20 hours. Then, 3.5 μL (10 vol-%) sodium acetate buffer (3.0 M NaOAc, pH 5.2) and 105 μL ethanol were added, and the mixture was incubated on ice for 2 hours. Next, the mixture was centrifuged (4°C, maximum speed, Centrifuge 5418R, Eppendorf), and the supernatant was discarded. The pellet was washed twice with 30 vol-% water containing 150 μL of ethanol. The supernatant was discarded, and the pellets were air-dried in a ventilated fume hood with the vials open for 20 minutes to minimize dust. The pellets were then redissolved in water. The nucleic acid sequences are summarized in Table VI, and the small molecule-nucleic acid conjugates are summarized in Table VII.

[0099] [Table 13]

[0100] [Table 14]

[0101] DNA conjugate extension. A DNA conjugate (20 μL, 50 μM (water); Conj_No_1, Conj_No_2 or Conj_No_3, SEQ ID NO. 16 (Microsynth) modified with 5'DBCO or SEQ ID NO. 17 (Microsynth) modified with 5'aminohexyl) was mixed with a split SEQ ID NO. 10 (20 μL, 100 μM (water), 2.0 equivalents). The 5' phosphorylated extender sequence of SEQ ID NO. 11 (30 μL, 100 μM (water), 3.0 equivalents, N-annotated degenerate nucleotide) in ligation buffer (10 μL, 10X, New England Biolabs, without ATP) and water (13.7 μL) was heated to 75°C and allowed to cool to room temperature over 1 hour. Next, ATP (10 μL, 10 mM (in water), 100 equivalents) and T4 DNA ligase (1.25 μL, 500 U, 1.2 U / pmol, New England Biolabs) were added and incubated at room temperature for 3 hours. Then, the solvent was removed, the residue was redissolved in water, and separated by HPLC (separation was performed on an Agilent 1260 Infinity II with a Concise RiboSep RNA column (PS / DVB-C18, non-porous, 7.8 × 50 mm), with a gradient of 1 mL / min from acetonitrile in 100 mM triethylammonium acetate in water at pH 7.2, at a temperature of 60°C (gradient: 5–8% in 1 min - 10.8% in 9 min - 25% in 7 min - 99% in 1 min (4 min) - 5% in 0.1 min)). The lyophyllated product was dissolved in 50 μL of water. The sequences of the starting materials are summarized in Table VIII. The yields and products are summarized in Table IX.

[0102] [Table 15]

[0103] [Table 16]

[0104] Proximity-induced extension of small molecule DNA conjugates with TdT fusion proteins. One of Conj_ID_No_4, Conj_ID_No_5, Conj_ID_No_6, or Conj_ID_No_8 (4 μL, 1 μM, 4 pmol) was mixed with sheared salmon sperm DNA (8 μL, 1 mg / mL), dATP (8 μL, 10 mM), and CAII-SUMO-TdTEvo (protein_SEQ ID NO: 7, 4 μL, 1 μM), and TdT buffer (80 μL, 10X, 500 mM KOAc, 200 mM TrisOAc, 100 mM MgOAc2, pH 7.9), and packed to a maximum volume of 800 μL. The mixture was incubated at 37°C for 30 minutes, then inactivated at 98°C for 10 minutes.

[0105] Poly-dT25 pulldown. Poly-dT 25 Magnetic beads (5 μL / pmol of the initial compound) were washed on a magnetic rack with pull-down buffer (20 μL, 20 mM Tris pH 7.5, 1 mM EDTA, 0.01% vol-% Tween 20) and resuspended in the same buffer (10 μL). Tween-20 (8 μL, 1 vol-%) was added to the inactivated sample for proximity-induced extension, and the resuspended beads were added. The sample was incubated at room temperature for 30 minutes, the beads were pulled down on a magnetic rack, and washed three times with pull-down buffer (100 μL). For elution, the beads were suspended in 40 μL of pull-down buffer, heated to 98°C over 10 minutes, and the supernatant was immediately collected and placed on the magnetic rack.

[0106] Urea PAGE analysis of closely spaced small molecule-DNA conjugates. A 1.5 mm gel was prepared by mixing acrylamide / bisacrylamide (1.25 mL, 40% 19:1, Roth) with urea (4.2 g, 7 M final concentration) and TBE (1 mL, 10X, 1 M Tris, 0.9 M boric acid, 0.01 M EDTA). The volume was adjusted to 10 mL, and ammonium persulfate (10 μL, 25%) and N-tetramethylethylenediamine (10 μL) were added. Three-quarters of the eluate from the previous step was mixed with formamide loading dye (10 μL, 2X, 95 vol-% formamide, 5 mM EDTA, 25 mg / L bromophenol blue, pH 8.0) and thermally denatured at 98°C for 5 minutes before loading onto the prepared gel. The gel was run in TBE buffer (1X) in a mini-PROTEAN system (BioRad) at a constant 120V until the blue band reached the bottom of the gel. The band was visualized by incubating the gel with SYBR gold (Thermo Fisher) according to the manufacturer's instructions.

[0107] qPCR analysis of closely spaced small molecule-DNA conjugates. 1 / 4 of the eluate was diluted to 1 / 1000, and 4 μL of this dilution was transferred to a 0.2 mL PCR tube. Specific primers for each conjugate (1 μL primer pair, 5 μM each) (see Table X for sequence list and Table XI for conjugate specifications) and 5 μL of PowerUP SYBR green master mix (ThermoFisher) were added. qPCR was performed using QuantStudio 1 with its standard protocol (program: 50°C (2 min) - 95°C (2 min) - (95°C (15 sec) - 57°C (15 sec) - 72°C (30 sec)) × 40 - 95°C (15 sec) - 60°C (1 min) - 95°C (15 sec)). ΔCT values ​​were converted to concentrations by measuring calibration curves from 1 nM to 10 fM for each conjugate. Each sample was subjected to three qPCR cycles.

[0108] [Table 17]

[0109] [Table 18]

[0110] Proximity-induced extension of single-stranded DNA encoding libraries with TdT fusion proteins. Single-stranded DNA encoding libraries synthesized according to the published procedure [5], having intact free 3' hydroxy ends (1.0 μL, 1.0 μM, 1.0 pmol, or 3.1e6 molecules per library member), were added to a mixture of sheared salmon sperm DNA (2 μL, 1 mg / mL), dATP (2 μL, 10 mM), and TdT buffer (20 μL, 10X, 500 mM KOAc, 200 mM TrisOAc, 100 mM MgOAc2, pH 7.9) to adjust the volume to 199 μL. Next, TdT fusions (SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 12; 1 μL, 1 μM) were added, and the mixture was incubated at 37°C for 30 minutes, then inactivated at 98°C for 10 minutes. As described above, dT 25 Bead pull-down was performed. For double-stranded DNA, blunt-ended DNA encoding libraries synthesized according to the published method [5], having free 3' hydroxyl groups and free 5' hydroxyl groups, were used, and the incubation time was extended to 1 hour instead.

[0111] Affinity enrichment of DNA-coding libraries. Nickel-NTA beads (20 μL, HisPur Ni-NTA magnetic beads, ThermoFisher) were washed three times with selective buffer (120 μL, Dulbecco's phosphate buffered saline with 0.01 vol-% Tween-20, 10 μg / mL sssDNA, 10 mM imidazole), and the supernatant was removed. The beads were then incubated at 4°C for 30 minutes in the presence or absence of protein (100 μL, 37 μM, Prot_SEQ ID NO: 10) or the absence of protein. DNA-coding libraries (1 μL, 1 μM, 1 pmol, single-stranded or double-stranded) and selective buffer (19 μL) were added to this volume and incubated at 25°C for 30 minutes. The beads were then washed five times with selective buffer (200 μL). The supernatant was removed, and the beads were resuspended in pull-down buffer (50 μL, 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.01 vol-% Tween-20) and incubated at 98°C for 10 minutes.

[0112] NGS library preparation. Based on the concentrations after selection determined by qPCR (1 μL sample, 2 μL water, 1 μL forward primer of SEQ ID NO: 25, 1 μL reverse primer of SEQ ID NO: 26 (5 μM each)) of the samples, 2 μL of each selection was used in a 50 μL PCR reaction with 10–22 cycles, keeping it within the linear amplification range (Phusion DNA polymerase, following the manufacturer's protocol (NEB), 30 seconds at 98°C, then the determined cycle lengths were 98°C (20 sec), 69°C (20 sec), 72°C (20 sec), and terminated at 72°C (300 sec) and 12°C (inf)). PCR was cleaned up using a PCR cleanup kit (Merchary & Nagel) according to its standard PCR purification protocol, except that the cutoff was adjusted by diluting NTI buffer with 5 volumes of water and two washes were performed. The cleaned-up PCR amplification products were eluted twice with 25 μL. 2 μL of these amplified products were used in a second PCR using index sequencing primers for the Illumina NGS platform (NEB, NebNext sets 1 and 2), with 1 μL of forward primer and 1 μL of reverse primer. Phusion DNA polymerase was used for 100 μL of reaction (98°C (30 sec), [98°C (20 sec), 69°C (20 sec), 72°C (20 sec)] × 15, 72°C (300 sec), 12°C (inf)). For each sample, an individual combination of indexes was used (manufacturer's protocol). All individual PCRs were pooled for equal concentrations (concentration was measured by visualizing bands on a urea PAGE gel by SYBR gold staining, but other methods are also usable) and sent for Illumina sequencing.

[0113] Data evaluation. Next-generation sequencing data were analyzed using DECL-Gen[5c] and individual scripts. In short, for each read, the unique molecular identifier (UMI) and the barcode (codon combination) encoding the compound were counted. For each UMI, only the most frequent codon combination was counted. Next, the number of all compounds in the selection containing the target protein was compared to the number of all compounds in the selection containing the matrix (SUMO-TdT in the case of TdT fusion proteins as described above, empty beads in the case of free proteins in the case of conventional affinity selection) according to the formula log2((x) / (y)) (where x is the mean number (normalized to 1 million reads) of the sample and y is the mean number (normalized to 1 million reads) of the control selection). The resulting number, called log-fold enrichment, was shifted to have a median of 1, and only compounds with a log-fold enrichment greater than 3 times the standard deviation of the log-fold enrichment were selected as promising binders.

[0114] [Table 19]

[0115] Further examples of fusion proteins. DNA sequences (Table XIII, SEQ ID NOs. 28, 30, and 42) or fragments thereof were purchased (AddGene or GeneScript) and cloned into the pET19b vector by restriction and ligation. The sequence of calmodulin was obtained from AddGene (182500) and encodes human CALM1 (P0DP23, SEQ ID NOs. 43). The sequence of SpyCatcher (SEQ ID NOs. 46) was obtained from a publicly available plasmid (AddGene, 133449) and synthesized BioCat. The sequence of SpyTag (SEQ ID NOs. 45) was obtained from AddGene (133450). Escherichia coli (E. coli) DH5α cells were transfected with the ligation product and spread on a plate containing antibiotics. Single colonies were sequenced, and positive clones were grown in LB medium. Plasmids were isolated and then transfected into Escherichia coli (E. coli) BL21 cells. Cells were grown at 37°C to an optical density of 0.6, then IPTG was added to a concentration of 0.5 mM, and the cell suspension was incubated overnight at 16°C. After cell lysis, proteins were bound to nickel beads, eluted with imizadole, and the buffer was replaced with a spin column. If necessary, the first SUMO tag and his tag were cleaved with TEV protease, and the protease was removed with nickel beads.

[0116] Conjugation of a calmodulin-peptide to DNA. A calmodulin-conjugated peptide (SEQ ID NO: 33) was synthesized by solid-phase peptide synthesis and capped with N-terminal 2-azidoacetic acid. The peptide was then incubated with DBCO-DNA (SEQ ID NO: 13) for 60 minutes and purified by elution using HPLC with a C4 column under an ammonium acetate (50 mM, pH 7) / 1% to 99% acetonitrile gradient. The pure fraction was lyophilized to obtain the pure product (calmodulin-peptide DNA conjugate).

[0117] Activity testing of Calmodulin-TdT. A Calmodulin-TdT (SEQ ID NO: 29) fusion protein containing Calmodulin (SEQ ID NO: 43), a linker (SEQ ID NO: 44), and TdTevo (SEQ ID NO: 5) was incubated with a Calmodulin-peptide DNA conjugate obtained under standard conditions. The polyadenylated product was pulled down using poly-T beads as described above. Gel (11% urea PAGE) was performed to determine the activity (Figure 6A) and selectivity (Figure 6B) of Calmodulin-TdT.

[0118] [Table 20]

[0119] [Table 21]

[0120] [Table 22]

[0121] [Table 23]

[0122] [Table 24]

[0123] [Table 25]

[0124] [Table 26]

[0125] Conjugation of BnG-DNA to SNAP-TdT. DNA-SNAP-TdT was prepared by incubating SNAP-SUMO-TdTEvo (1 μL, 157 ng μL-1 (in storage buffer), 2 pmol, 1 equivalent, SEQ ID NO: 12) with BnG-modified DNA (2 μL, 1 μM (in H2O), 2 pmol, 1 equivalent, SEQ ID NO: 34) at 25°C for 15 minutes (see Figure 7A). A new coupling was performed before each assay and individually for each experiment (no bulk). In the case of untemplated assays, SNAP-SUMO-TdTEvo was incubated under the same conditions as H2O. Table XIV shows the DNA dissociation constants for SEQ ID NO: 34 calculated by the nearest neighbor method. [BnG] is named the benzoguanine portion, and [FAM] is named the fluorescein (FAM) portion.

[0126] [Table 27]

[0127] [Table 28]

[0128] Extension of SpyTag-CAII and SpyCatcher-TdT. 8 μL of SpyCatcher-SUMO-TdT (6 μM, SEQ ID NO: 32) and 1.8 μL of SpyTag-CAII (30 μM, SEQ ID NO: 41) were mixed and incubated on ice for 20 minutes. Next, a TdT extension assay was set up on a 4x larger scale (800 μL) using standard conditions without sssDNA to improve gel readout. Conjugates 1-3 (Table VII) were used as substrates for extension. After TdT inactivation, the product was concentrated using dT25 beads as described above and eluted on an 11% urea PAGE gel. Figure 6C shows the extension of the DNA small molecule conjugate along with the Spytag-CAII-SpyCatcher-TdT conjugate.

[0129] Loading of multiple TdT fusion proteins onto POI. POI (10 μL, 30 μM) was incubated with BG-maleimide (10 μL, 1 mM) in PBS buffer at room temperature for 2 hours and purified by spin column. Subsequently, the protein (10 μL, 3 μM) was incubated with Snap-TdT (SEQ ID NO: 12) (10 μL, 9 μM) at room temperature for 2 hours to obtain an average of X TdTs / POI.

[0130] B) Result Proximity-induced elongation of small molecule DNA conjugates. Figure 1B shows the elongation of individual small molecule DNA conjugates by the CAII-TdT fusion protein. As affinity increases, the conjugate DNA strand is elongated more (marked by gray bars). The darker areas, particularly in the uppermost gel, are due to either nonspecific binding of sheared salmon sperm DNA or elongation in the background. Conjugates containing only amines do not show bands for enrichment and elongation.

[0131] qPCR analysis of closely spaced small molecule-DNA conjugates. Figure 2 shows the same experiment as Figure 1B, but instead quantitatively measured by qPCR. Furthermore, the amount of small molecule DNA conjugate is measured instead of the amount and distribution of the poly(A) tail. All three binders are enriched to at least 30-fold above the background conjugate.

[0132] Proximity-induced elongation DNA bound to a benzoguanine-DNA conjugate conjugated with SNAP. Figure 7 shows a model system where the affinity of small molecule-DNA conjugates to POIs is simulated with DNA-DNA pairs of different affinities. Conjugates of Snap-TdT (SEQ ID NO: 12) and BG-DNA (SEQ ID NO: 34) are mixed with DNA of various affinities shown above. (Affinity for SEQ ID NO: 34 decreases from SEQ ID NO: 36 to SEQ ID NO: 40). As affinity increases, more dATP is incorporated into the DNA strand, resulting in a longer DNA tail. When DNA is not conjugated with SNAP-TdT, the elongation of the substrate DNA (SEQ ID NO: 36) is minimal.

[0133] Affinity enrichment of DNA-coding libraries. Comparing the number of compounds selected from single-stranded DNA-coding libraries for His-CAII (SEQ ID NO: 10) to the selection for empty beads, 143 compounds (73%) were recovered from 192 containing acetazolamide (Figure 3A, solid arrow) and 979 compounds (38%, dotted arrow) were recovered from 2606 containing phenylsulfonamide. For chemically identical double-stranded DNA-coding libraries, 157 acetazolamide compounds (82%, Figure 3B, solid arrow) were found from 192 and 1158 phenylsulfonamide compounds (44%, dotted arrow) were found from 2606.

[0134] Proximity-induced extension of DNA-coding libraries. Comparing the number of compounds selected from a single-stranded DNA-coding library for the CAII-SUMO-TdT fusion protein (SEQ ID NO: 11) with the number of selections for the SUMO-TdT fusion protein (SEQ ID NO: 7), 145 out of 192 compounds containing acetazolamide (74%) (Figure 4A, solid arrow) and 2319 out of 2606 compounds containing phenylsulfonamide (89%, Figure 4A, dotted arrow) were recovered (significantly more than observed in affinity enrichment). Furthermore, the inventors identified 21 compounds containing the N-methoxyphenylsulfonamide motif (11%, dotted arrow). When incubated with half the amount of protein, 151 acetazolamide-containing compounds were recovered (79%, Figure 4B), but the log-fold value was higher. Additionally, 2243 phenylsulfonamide compounds were found (86%). The N-methoxyphenylsulfonamide motif was found 20 times more frequently (10%). In the case of double-stranded DNA encoding libraries, 126 acetazolamides (66%, Figure 5A) and 1843 phenylsulfonamides (71%) were found. In the case of half the amount of protein, 115 acetazolamides were found (60%, Figure 5B), but the log-fold enrichment was higher, and 1419 phenylsulfonamides were found (54%).

[0135] Activity testing of calmodulin-TdT and proximity-induced extension of a binder-DNA conjugate. Calmodulin-TdT (SEQ ID NO: 29) is active (Figure 6A) and can extend the 3' end of a given DNA via dATP. DNA modified with calmodulin-peptide binder (SEQ ID NO: 33) extends more than DNA conjugated with DBCO small molecules alone (Figure 6B), demonstrating an induced proximity effect of the system.

[0136] Proximity induction of the CAII-SpyTag-SpyCatcher-SUMO-TdT conjugate. Figure 6C shows the formation of the SpyTag-CAII (37kDa) and SpyCatcher-SUMO-TdT (92kDa) conjugates. When both are present, a new band is formed at approximately 130kDa as a conjugation between SpyCatcher and SpyTag. Figure 6D shows the proximity-induced extension of conjugates 1-3 (Table VII) by SpyTag-CAII-SpyCatcher-TdT. The unbound conjugate (SEQ ID NO: 16) does not show proximity extension.

[0137] C) Summary Proximity-induced poly(A) tailing by CAII-TdT fusion proteins and subsequent enrichment of the extended tail are similarly effective in recovering binders with KDs in the double-digit nanomolar range, as shown in Figures 3–5. In the case of single-stranded DNA encoding libraries, 74–79% of these binders were recovered, compared to 73% recovered by conventional affinity enrichment methods. Surprisingly, the present invention is significantly superior to state-of-the-art affinity enrichment methods when recovering micromolar binders [7]. Nearly 90% of phenylsulfonamids were found to be present in the library, compared to only 38% with state-of-the-art affinity enrichment methods. Furthermore, the present invention identified approximately 20–21 (10–11%) of N-methoxyphenylsulfonamides, known as very weak inhibitors of CAII with Kis almost an order of magnitude lower than those of non-methoxylated analogs, in amounts of 192 [7]. This motif is completely invisible in state-of-the-art affinity enrichment and is exclusively captured by the present invention. This makes the method of the present invention a complete screening tool for proteins that are difficult to target.

[0138] This method further identifies more than two-thirds (66%, 82% in affinity enrichment) of strong binders in double-stranded DNA encoding libraries. 71% of weak binders are identified. Although this is less than what is obtained by methods using single-stranded libraries, it is still significantly higher than in state-of-the-art affinity enrichment methods (44%) [7].

[0139] Furthermore, this method works in conjunction with sub-nanomolecular peptide-protein interactions, as seen in proximity-induced extension of calmodulin-bound peptide-DNA conjugates and calmodulin-TdT. Proximity-induced extension also works when artificial conjugates are created between the target protein and TdT using SpyTag-SpyCatcher technology.

[0140] List of References [1] Barthel, S., Palluk, S., Hillson, NJ, Keasling, JD & Arlow, DH “Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis”.Genes 11,102(2020). [2] More, KNet al. “Acetazolamide-based [ 18 F]-PET tracer: In vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressingHypoxic solid tumors”.Bioorganic & Medicinal Chemistry Letters 28,915-921(2018). [3] Katzl,K.& Ruis,H.《Ueber tricyclische 1,2,4-Benzothiadiazine》.Monatshefte fuer Chemie 96,1603-1610(1965). [4] Akocak,2016,“PEGylated Bis-Sulfonamide Carbonic Anhydrase Inhibitors Can Efficiently Control the Growth of Several Carbonic Anhydrase IX-Expressing Carcinomas”.Journal of Medicinal Chemistry 59,5077-5088(2016). [5](a)Stress,C.,Sauter,B.,Schneider,L.,Sharpe,T.& Gillingham,D.“A DNA-encoded chemical library incorporating elements of natural macrocycles”.Angew.Chem.Int.Ed.58,9570-9574(2019).(b)Bassi,G.et al.“A Single-Stranded DNA-Encoded Chemical Library Based on a Stereoisomeric Scaffold Enables Ligand Discovery by Modular Assembly of Building Blocks”.Advanced Science 7,2001970(2020).(c)Sauter,B.Doctoral Thesis,“Applications of Next Generation Sequencing in the Field of Chemical Biology”.(University of Basel,2020)doi:10.5451 / UNIBAS-EP88004. [6](a)Wichert,M.et al.《Dual-display of small molecules enables the discovery of ligand pairs and facilitates affinity maturation.” Nature Chemistry 7,241-249(2015).(b)Kazmierski,W.M.et al.“DNA-Encoded Library Technology-Based Discovery,Lead Optimization,and Prodrug Strategy toward Structurally Unique Indoleamine 2,3-Dioxygenase-1(IDO1)Inhibitors.” Journal of Medicinal Chemistry 63,3552-3562(2020).(c)Stress,C.,Sauter,B.,Schneider,L.,Sharpe,T.& Gillingham,D.“A DNA-encoded chemical library incorporating elements of natural macrocycles.” Angew.Chem.Int.Ed.58,9570-9574(2019). [7](a)Briganti,F.,Pierattelli,R.,Scozzafava,A.& Supuran,C.Carbonic anhydrase inhibitors.Part 37.Novel classes of isozyme I and II inhibitors and their mechanism of action.Kinetic and spectroscopic investigations on native and cobalt-substituted enzymes.European Journal of Medicinal Chemistry 31,1001-1010(1996).(b)Fiore,A.D.,Maresca,A.,Alterio,V.,Supuran,C.T.& Simone,G.D.Carbonic anhydrase inhibitors:X-ray crystallographic studies for the binding of N-substituted benzenesulfonamides toHuman isoform II.Chemical Communications 47,11636(2011). [8] Keeble A.H.et al.“Approaching infinite affinity through engineering of peptide-protein interaction” PNAS,116,26523-26533(2019).

Claims

1. A fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof, provided that it is not a fusion protein containing the terminal deoxynucleotidyltransferase (TDT) and Cas9.

2. The fusion protein according to claim 1, comprising a linker between a DNA polymerase or fragment thereof having terminal transferase activity and the target protein (POI) or fragment thereof.

3. The fusion protein according to claim 1 or 2, wherein the DNA polymerase or fragment thereof having terminal transferase activity is terminal deoxynucleotidyltransferase (TDT) or fragment thereof.

4. The fusion protein according to claim 3, wherein the terminal deoxynucleotidyltransferase (TDT) or a fragment thereof comprises the sequence shown in SEQ ID NO:

5.

5. The fusion protein according to any one of claims 1 to 4, wherein the POI or fragment thereof is selected from the group consisting of an enzyme or fragment thereof and a protein or fragment thereof that targets or is involved in cell proliferation.

6. A fusion protein according to any one of claims 1 to 4, comprising a sequence selected from the group shown by SEQ ID NOs: 7 to 12.

7. A fusion protein according to any one of claims 1 to 6, which is bound to a conjugate compound comprising a binder for POI or a fragment thereof and a nucleic acid moiety.

8. The fusion protein according to claim 7, wherein the nucleic acid portion is ssDNA or dsDNA.

9. A fusion protein according to any one of claims 1 to 8, comprising a DNA polymerase or fragment thereof having terminal transferase activity and a calmodulin or fragment thereof.

10. A fusion protein according to any one of claims 1 to 9, comprising a DNA polymerase or fragment thereof having terminal transferase activity, and two or more copies of the target protein (POI) or two or more copies of a fragment thereof.

11. A fusion protein according to any one of claims 1 to 9, comprising two or more copies of a DNA polymerase having terminal transferase activity or two or more copies of a fragment thereof, and a target protein (POI) or a fragment thereof.

12. A fusion protein according to any one of claims 1 to 9, comprising two or more copies of a DNA polymerase having terminal transferase activity or two or more copies of a fragment thereof, and two or more copies of the target protein (POI) or two or more copies of a fragment thereof.

13. The fusion protein according to any one of claims 1 to 12, comprising one or more linkers between a DNA polymerase or fragment thereof having terminal transferase activity and the target protein (POI) or fragment thereof, wherein the linkers are chemical entities, and each of the one or more linkers is fused in part to an amino acid of the target protein (POI) or fragment thereof that is different from the N-terminal and / or C-terminal amino acid of the target protein (POI) or fragment thereof, and each of the same one or more linkers is fused in another part of the linker to a DNA polymerase or fragment thereof having terminal transferase activity.

14. A fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and calmodulin or fragment thereof, which is bound to a further fusion protein comprising a calmodulin-binding peptide and POI or fragment thereof.

15. A method for screening molecules for binding to a target protein (POI) or a fragment thereof, a) i) prepare an incubation medium containing deoxynucleotide triphosphate (dNTPs), ii) a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and the protein of interest (POI) or fragment thereof, and iii) a conjugate compound comprising a molecule and a nucleic acid portion; and incubation the fusion protein and the conjugate compound in the incubation medium; b) Separating the conjugate compound prepared in step a) in which the nucleic acid portion is extended from the conjugate compound prepared in step a) in which the nucleic acid portion is not extended; c) a step of amplifying and analyzing the extended nucleic acid portion of the conjugate compound obtained in step b) and prepared in step a); d) A step of correlating the extended nucleic acid portion analyzed in step c) with the corresponding molecule of the conjugate compound used in step a), A method comprising the molecule correlated in step d) being selected as the binder for the POI.

16. A method for screening molecules according to claim 15, wherein in step a), the DNA polymerase or fragment thereof having terminal transferase activity is inactivated before step b) is carried out.

17. Step b) is a method for screening molecules according to claim 15 or 16, comprising adding a solid phase containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound to the incubation medium, and separating the solid phase containing the deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound, which is hybridized to the extended nucleic acid portion of the conjugate compound, from the solid phase containing the deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound, which is not hybridized to the extended nucleic acid portion of the conjugate compound.

18. A method for screening molecules according to claim 17, wherein the conjugate compound having the extended nucleic acid portion is eluted from a solid phase containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound.

19. A method for screening molecules according to claim 18, wherein the washing step is performed after the conjugate compound having the extended nucleic acid portion has been eluted from the solid phase containing a deoxynucleotide complementary to the extended nucleic acid portion of the conjugate compound.

20. A method for screening molecules according to any one of claims 15 to 19, wherein the extended nucleic acid portion is amplified and sequenced in step c).

21. A method for screening a molecule according to any one of claims 15 to 20, wherein the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof is the fusion protein according to any one of claims 1 to 8.

22. A method for screening a molecule according to any one of claims 15 to 20, wherein the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a target protein (POI) or fragment thereof is the fusion protein according to any one of claims 1 to 14.

23. A method for screening a molecular DNA-coding library for binding to a target protein (POI) or a fragment thereof, a) i) an incubation medium containing deoxynucleotide triphosphate (dNTP), ii) a fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof, and iii) a DNA-coding library of chemical molecules, comprising multiple entities of a single molecule of the library, each entity being covalently bonded to a nucleic acid portion to form a conjugate compound comprising the molecule and the nucleic acid portion; a step of incubating the fusion protein and the conjugate compound; b) Separating the conjugate compound prepared in step a) in which the nucleic acid portion is extended from the conjugate compound prepared in step a) in which the nucleic acid portion is not extended; c) a step of amplifying and analyzing the extended nucleic acid portion of the conjugate compound obtained in step b) and prepared in step a); d) Correlating the extended nucleic acid portion analyzed in step c) with the corresponding plurality of entities of one single chemical molecule in the library used in step a), A method comprising, wherein the plurality of entities of one single chemical molecule of the library thus correlated in step d) are selected as binders for the POI.

24. A method for screening a DNA encoding library of a molecule according to claim 23, wherein the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof is the fusion protein according to any one of claims 1 to 8.

25. A method for screening a DNA encoding library of a molecule according to claim 23, wherein the fusion protein comprising a DNA polymerase or fragment thereof having terminal transferase activity and a protein of interest (POI) or fragment thereof is the fusion protein according to any one of claims 1 to 14.

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