KR20210090010A - An L-DNA-binding natural protein and uses thereof - Google Patents

Abstract

The present invention relates to a protein including a domain exhibiting a property of binding with L-DNA and uses thereof, and more particularly, to an isolated L-DNA binding domain including an amino acid sequence represented by SEQ ID NO: 1, a protein including the isolated L-DNA binding domain, a drug carrier including the protein, and a pharmaceutical composition for the treatment of cancer including the drug carrier and an anticancer agent. The protein including the isolated L-DNA binding domain according to the present invention can be applied to the preparation of antibody-drug conjugates to deliver drugs in a target-selective manner. In addition, when the protein containing the isolated L-DNA binding domain is applied to the conjugation of the antibody and the drug, it is possible to label a drug as much as a desired amount in a site-selective manner without interfering with a target recognition site of the antibody for delivering the drug to a specific target.

Description

Natural protein binding to L-DNA and uses thereof {An L-DNA-binding natural protein and uses thereof}

The present invention relates to a protein comprising a domain exhibiting binding properties with L-DNA and uses thereof. More specifically, an isolated L-DNA binding domain comprising the amino acid sequence represented by SEQ ID NO: 1, a protein comprising the isolated L-DNA binding domain, a drug carrier comprising the protein, and the drug carrier and an anticancer agent It relates to a pharmaceutical composition for the treatment of cancer comprising a.

L-DNA is the enantiomer of natural D-DNA. Due to the mirror image structure, L-DNA does not hybridize with D-DNA (BE Young. et.al. , Chemistry - A European Journal , 25:7981-7990, 2019). Although L-DNA is not used in natural genetic systems, this bioorthogonal base-pairing system has been mainly applied in biotechnology to improve the target specificity of gene detection systems (G. Ke. et.al. , Journal of the American Chemical Society , 134:18908-18911, 2012; NC Hauser et.al. , Nucleic acids research , 34:5101-5111, 2006). The chirality of L-DNA is incompatible with naturally occurring DNA modifying enzymes including polymerases, ligases and nucleases. In particular, the nuclease resistance of L-DNA has been used to secure the stability of aptamers in a biological environment (KP Williams et.al. , Proceedings of the National Academy of Sciences , 94:11285-11290, 1997). In addition, as the L-DNA duplex maintains the same thermodynamic properties as the natural DNA duplex (WG Purschke et.al. , Nucleic acids research , 31:3027-3032, 2003), the D-DNA nanostructures were Various self-assembled L-DNA nanostructures could be easily prepared by borrowing the sequences previously used for construction (K.-R. Kim et.al. , Journal of Controlled Release , 243:121-131). , 2016; C. Lin et. al. , Nano Letters , 9:433-436, 2009). L-DNA nanostructures showed not only improved serum stability but also relatively high cellular uptake efficiency and similar biocompatibility compared to their natural counterpart (K.-R. Kim et.al. , Chemical Science , 5: 1533-1537, 2014). These properties of L-DNA nanostructures have been successfully utilized to develop drug delivery platforms for in vivo applications (K.-R. Kim et.al. , Biomaterials , 195:1-12, 2019; AY Lee et. al. , Journal of Industrial and Engineering Chemistry , 74:187-192, 2019).

In order to deliver protein drugs using L-DNA nanostructures, conjugation between L-DNA and proteins is required. For this purpose, an easily available method is to connect the L-DNA end to a carboxylic acid with easy chemical bonding. (carboxylic acid), amine (amine), or thiol (thiol) functional group is transformed, and it sends a cross-linking reaction with the amine functional group of lysine (Lys) of the protein. However, this method has a disadvantage in that it is difficult to selectively bind L-DNA to only a specific Lys among several Lys in a protein. If there is a domain that can bind to L-DNA among the domains of the protein, when the L-DNA is mixed with the L-DNA binding domain into the protein drug to be loaded, the protein drug is naturally added to the L-DNA. It can be combined and mounted. In addition, recently, in order to effectively deliver gene drugs targeting disease genes such as siRNA or antisense oligonucleotides to disease sites, antibody-oligonucleotide conjugates are used by binding an antibody to an oligonucleotide gene drug. , attempts to utilize AOCs) are active. In this case, it may be possible to bind the gene drug to a specific part of the antibody by introducing the L-DNA binding domain into the antibody and extending the end of the gene drug to L-DNA to bind to each other. However, among the proteins known in nature, there has been no report of a protein having a domain capable of binding to L-DNA so far. Structural information to determine the interaction between L-DNA and a natural protein is essential to determine whether it can have binding to L-DNA. This information also provides information on the interaction between natural DNA, D-DNA and protein. Information is very scarce.

Contrary to the number of studies on the structure of various natural DNA forms and their complexes with proteins (TA Steitz, Journal of Biological Chemistry , 274:17395-17398, 1999; PJ Paukstelis et.al. , Crystals , 6:97, 2016) ; NM Luscombe et.al. , Genome Biology , 1, reviews001.1., 2000), studies on the structure of L-DNA were very limited. Structural insight into native DNA is not surprising as it provides more useful information for understanding many DNA-related biological functions. Nevertheless, as many functional L-DNA structures begin to emerge, the structural description of L-DNA structures and their complexes with proteins at the molecular level is important in understanding how mirror DNA can be integrated into biological systems, and L-DNA It is important to come up with a way to design new enzymes that work in Previously, the crystal structure of an L-DNA duplex was reported in a racemic mixture (PK Mandal et.al. , Angewandte Chemie International Edition , 53:14424-14427, 2014). The determination of the Holliday junction of the self-assembled L-DNA trigonal system was also obtained very recently (CR Simmons et.al. , Journal of the American Chemical Society , 139:11254-11260, 2017). In terms of the structure of L-DNA/protein complexes, very few cases have been reported since L-DNA-protein interactions are not freely available in nature. The only known crystal structure of a mirror DNA/native protein complex describes the interaction between a native protein and its experimentally developed mirror DNA aptamer (or DNA spiegelmer) (L. Yatime et.al. , Nature Communications , 6:6481, 2015). However, the intrinsic L-DNA binding properties of native proteins have not been explored so far.

B. E. Young. et.al., Chemistry - A European Journal, 25:7981-7990, 2019 G. Ke. et.al., Journal of the American Chemical Society, 134:18908-18911, 2012 N. C. Hauser et. al., Nucleic acids research, 34:5101-5111, 2006 K. P. Williams et. al., Proceedings of the National Academy of Sciences, 94:11285-11290, 1997 W. G. Purschke et.al., Nucleic acids research, 31:3027-3032, 2003 K.-R. Kim et.al., Journal of Controlled Release, 243:121-131, 2016 C. Lin et. al., Nano Letters, 9:433-436, 2009 K.-R. Kim et.al., Chemical Science, 5:1533-1537, 2014 K.-R. Kim et.al., Biomaterials, 195:1-12, 2019 A. Y. Lee et.al., Journal of Industrial and Engineering Chemistry, 74:187-192, 2019 T. A. Steitz, Journal of Biological Chemistry, 274:17395-17398, 1999 P. J. Paukstelis et. al., Crystals, 6:97, 2016 N. M. Luscombe et.al., Genome Biology, 1, reviews001.1., 2000 P. K. Mandal et. al., Angewandte Chemie International Edition, 53:14424-14427, 2014 C. R. Simmons et.al., Journal of the American Chemical Society, 139:11254-11260, 2017 L. Yatime et. al., Nature Communications, 6:6481, 2015

Under the above background, the present inventors made diligent efforts to develop a novel biocompatible drug delivery system. As a result, L-DNA, which is a mirror image of natural DNA, and the LF domain derived from DNA polymerase Dpo4 (little figner domain) are combined By revealing the characteristics, the present invention was completed.

It is an object of the present invention to provide a protein domain having binding affinity to L-DNA.

Another object of the present invention is to provide an isolated L-DNA binding domain comprising the amino acid sequence represented by SEQ ID NO: 1 and a protein comprising the isolated L-DNA binding domain.

Another object of the present invention is to provide a drug delivery system comprising a protein comprising the isolated L-DNA binding domain.

Another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising the drug carrier and an anticancer agent.

The object of the present invention is not limited to the object mentioned above. The objects of the present invention will become more apparent from the following description, and will be realized by means and combinations thereof described in the claims.

In order to achieve the above object, the following solutions are provided.

One aspect of the present invention provides a protein domain having binding affinity to L-DNA.

In one aspect of the present invention, the domain provides a protein domain having 30% or more sequence homology with SEQ ID NO: 2.

In one aspect of the present invention, the domain provides a protein domain having at least 80% sequence homology with SEQ ID NO: 2.

Another aspect of the present invention provides an isolated L-DNA binding domain comprising the amino acid sequence represented by SEQ ID NO: 1.

In another aspect of the present invention, there is provided an isolated L-DNA binding domain, wherein the isolated L-DNA binding domain is derived from a DNA polymerase.

In another aspect of the present invention, the DNA polymerase provides an isolated L-DNA binding domain, which is DNA polymerase 4 (Dpo4).

In another aspect of the present invention, there is provided an isolated L-DNA binding domain, wherein the isolated L-DNA binding domain is a LF domain (little figner domain) of a DNA polymerase.

In another aspect of the present invention, the isolated L-DNA binding domain is from the group consisting of Arg (Arginine) at position 300, Lys (Lysine) at position 321, and Lys (Lysine) at position 339 of the amino acid sequence shown in SEQ ID NO: 2 and wherein at least one selected amino acid residue is unsubstituted or mutated.

Another aspect of the present invention provides a protein comprising the domain.

In another aspect of the present invention, the protein is applied to the preparation of antibody-drug conjugates (antibody-drug conjugates), it provides a protein.

In another aspect of the present invention, the drug is selected from the group consisting of anticancer drugs, aptamers, antisense oligonucleotides, small interfering ribonucleic acids (siRNA, small interfering RNA) and micro RNA (miRNA), providing a protein do.

Another aspect of the present invention provides a drug delivery system comprising the protein.

Another aspect of the present invention is the drug delivery system; And it provides a pharmaceutical composition for the treatment of cancer, including an anticancer agent.

In another aspect of the present invention, the anticancer agent is a DNA aptamer or an RNA aptamer, it provides a pharmaceutical composition.

A protein comprising an isolated L-DNA binding domain, consisting of the amino acid sequence represented by SEQ ID NO: 1 according to an aspect of the present invention, is applied to the preparation of antibody-drug complexes (antibody-drug conjugates) to selectively deliver drugs can have an effect.

In addition, when the protein comprising the isolated L-DNA binding domain according to one aspect of the present invention is applied to the conjugation of an antibody and a drug, it interferes with the target recognition site of the antibody for delivering the drug to a specific target Since it is possible to label a drug as much as a desired quantity in a site-selective manner, it can be usefully used for preparing an antibody-drug complex.

The effects of the present invention are not limited to the above-mentioned effects. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

Figure 1 shows that native DNA polymerase binds to D-DNA using pockets formed by key domains such as thumb, palm and finger (top), and DNA polymerase is L-DNA It is a diagram schematically showing whether it is not known whether it can bind to (bottom).
2 is a diagram showing the results of 6% Native PAGE screening for polymerase binding to D-DNA (top) and L-DNA (bottom). P and T represent primer and template, respectively.
3 is a diagram showing the L-DNA binding properties of Dpo4 (DNA polymerase 4). Specifically, (a) is a diagram showing the DNA binding affinity of Dpo4 estimated by FP analysis. The concentration of fluorescently labeled DNA was 100 nM. (b) A diagram showing competition between D-DNA and L-DNA for Dpo4 binding. D-DNA was titrated with a solution of Dpo4 (1 μM) previously incubated with 100 nM of fluorescently labeled L-DNA. (c) A diagram showing the results of 20% denaturation PAGE analysis of primer extension reactions for fluorescently labeled D-primer and L-primer (100 nM). P and P+1 denote primers and single nucleotide-extended primers, respectively. (d) A diagram showing the measurement _{of K d} values of Dpo4 binding to various DNA duplexes with different GC-contents (100 nM) using FP analysis. GC-0, GC-33, GC-67 and GC-100 show 0%, 33%, 67% and 100% GC content in the DNA duplex, respectively.
4 is a diagram showing the crystal structure of Dpo4 forming a complex with D-DNA and L-DNA. Specifically, (a) is a diagram showing the overall structure of the Dpo4/D-DNA binary complex. (b) A diagram showing the overall structure of the Dpo4/L-DNA binary complex. (c) A diagram showing overlapping Dpo4 complexed with apo-Dpo4 (pink), D-DNA (turquoise) and L-DNA (light blue). Structural alignments of the amino acid portion of the Dpo4/D-DNA complex, the Dpo4/L-DNA complex, and apo-Dpo4 are shown. The overall Dpo4 structure (top) is shown, with a magnified view of the amino acid residues around the flexible linker between the little finger and other core domains, the catalytic core domain (bottom left), and the little finger domain (bottom right). The C-alpha atoms of the Dpo4/D-DNA duplex and the Dpo4/L-DNA duplex were aligned with the C-alpha atom of the apo-Dpo4 structure with the PyMol (PDB: 2RDI) program. (d) A diagram showing the structure of the dimerized little finger domain in the Dpo4/L-DNA complex and a 90° rotation view thereof. (e) A diagram showing the estimated binding stoichiometry curve using an isothermal titration calorimeter.
5 is a diagram suggesting an important role of a little finger (LF) in Dpo4 for interaction with L-DNA. (a) The binding affinity of Dpo4, little finger cleaved Dpo4 (Dpo4-ΔLF), little finger mutated Dpo4 (Dpo4-mutLF) and little finger domain (LF) with L-DNA was measured using fluorescence polarization analysis. A diagram showing a result. (b) A diagram showing amino acid residues from the little finger of the chain A responsible for interaction with the minor groove and the chain B responsible for the interaction with the main groove of the L-DNA duplex. (c) A diagram showing a schematic diagram of the Dpo4-L-DNA interaction.
Figure 6 is (a) 6% Native PAGE results and (b) SigmaPlot software (SigmaPlot software; Systat, USA) performed to estimate the degree of binding of L-DNA to various concentration conditions of Dpo4 from various archaea species. ) is a diagram showing the _{K d} value determined by quantification of band intensity and nonlinear regression for a four-parameter logistic curve equation.
7 is a drug delivery principle of antibody-drug complexes (antibody-drug conjugates) prepared by applying a protein comprising an isolated L-DNA binding domain, consisting of the amino acid sequence represented by SEQ ID NO: 1 according to the present invention; is a diagram shown as
8 is a S. solfataricus (S. solfataricus: Ss) Dpo4 represented by SEQ ID NO: 2 according to the present invention, as shown in SEQ ID NO: 11 Sulfolobus acidocaldarius (Sulfolobus acidocaldarius: Sa) Dpo4 It is a diagram confirming the homology (55.6%) by sorting.
9 is a S. solfataricus (S. solfataricus: Ss) Dpo4 represented by SEQ ID NO: 2 according to the present invention, the Picrophilus torridus represented by SEQ ID NO: 12 ( Picrophilus torridus: Pt) Dpo4 It is a diagram confirming homology (48.2%) by sorting.
10 is a metallosphaera sedula (Metallosphaera sedula : Ms) Dpo4 represented by SEQ ID NO: 13 based on S. solfataricus (Ss) Dpo4 represented by SEQ ID NO: 2 according to the present invention; It is a diagram confirming homology (56.8%) by sorting.
11 is a nitrosospira represented by SEQ ID NO: 14 based on S. solfataricus (Ss) Dpo4 represented by SEQ ID NO: 2 according to the present invention; Vienensis ( Nitrososphaera viennensis : Nv) is a diagram confirming homology (35.7%) by aligning Dpo4.
12 is an acidiplasma aeolicum (Acidiplasma aeolicum: Aa) Dpo4 represented by SEQ ID NO: 15 based on S. solfataricus (Ss) Dpo4 represented by SEQ ID NO: 2 according to the present invention; It is a diagram confirming homology (44.5%) by sorting.
13 is a view showing five different species in the archaeal domain based on S. solfataricus : Ss Dpo4 represented by SEQ ID NO: 2 according to the present invention ( Sulfolobus acidocaldarius [Sulfolobus acidocaldarius: Sa (SEQ ID NO: 11)], Picrophilus torridus [Picrophilus torridus: Pt (SEQ ID NO: 12)], Metallosphaera sedula [Metallosphaera sedula: Ms (SEQ ID NO: 13)], nitrosospira Dpo4 from Vienensis [Nitrososphaera viennensis: Nv (SEQ ID NO: 14)], Acidiplasma aeolicum [Acidiplasma aeolicum: Aa (SEQ ID NO: 15)]) using the EMBOSS Needle program, is a diagram showing alignment .

Unless otherwise specified, all numbers, values, and/or expressions expressing ingredients, reaction conditions, and amounts of ingredients used herein refer to a variety of measures that may occur in obtaining such values, among others, in which such numbers are inherently different. Since they are approximations reflecting uncertainty, it should be understood as being modified by the term "about" in all cases. Also, where the disclosure discloses numerical ranges, such ranges are continuous and inclusive of all values from the minimum to the maximum inclusive of the range, unless otherwise indicated. Furthermore, when such ranges refer to integers, all integers inclusive from the minimum to the maximum inclusive are included, unless otherwise indicated.

In this specification, when a range is described for a variable, the variable will be understood to include all values within the stated range including the stated endpoints of the range. For example, a range of “5 to 10” includes the values of 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. It will be understood to include any value between integers that are appropriate for the scope of the recited range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like. Also for example, a range of "10% to 30%" includes values such as 10%, 11%, 12%, 13%, and all integers up to and including 30%, as well as 10% to 15%, 12% to It will be understood to include any subrange, such as 18%, 20% to 30%, etc., as well as any value between reasonable integers within the scope of the recited range, such as 10.5%, 15.5%, 25.5%, and the like.

Hereinafter, the present invention will be specifically described.

The present invention relates to a protein comprising a domain exhibiting binding properties with L-DNA, which can be applied to the preparation of antibody-drug conjugates to selectively deliver a drug to a target, and uses thereof. More specifically, an isolated L-DNA binding domain comprising the amino acid sequence represented by SEQ ID NO: 1, a protein comprising the isolated L-DNA binding domain, a drug carrier comprising the protein, and the drug carrier and an anticancer agent It relates to a pharmaceutical composition for the treatment of cancer comprising a.

One aspect of the present invention provides a protein domain having binding affinity to L-DNA.

In the present invention, the term “L-DNA” is an enantiomer of natural D-DNA, and due to the mirror image structure, L-DNA does not hybridize with D-DNA. It is known that the chirality of L-DNA is incompatible with naturally occurring DNA modifying enzymes including polymerases, ligases and nucleases. In particular, the nuclease resistance of L-DNA has been used to secure the stability of aptamers in a biological environment. In addition, various self-assembled L-DNA by borrowing sequences previously used to construct D-DNA nanostructures, as L-DNA duplexes retain the same thermodynamic properties as native DNA duplexes. It has been reported that nanostructures can be easily prepared. L-DNA nanostructures show not only improved serum stability, but also relatively high cellular uptake efficiency and similar biocompatibility compared to their natural counterparts, and these properties of L-DNA nanostructures are useful for drug delivery for in vivo applications. It has been reported that it can be used to develop a platform.

As used herein, the term "protein domain" refers to a polypeptide that binds to L-DNA in vivo or in vitro. The 'polypeptide' is an amino acid polymer formed by peptide bonds of 10 or more amino acids, and is not limited to any specific polypeptide as long as it can bind to L-DNA.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

In the present invention, the protein domain is not limited thereto, but the sequence homology with SEQ ID NO: 2 is 30%, 40%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 86 contains an amino acid sequence that is at least %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical It may be a protein domain having binding affinity to L-DNA.

In one embodiment of the present invention, in order to investigate the L-DNA binding ability of Dpo4 derived from Sulfolobus solfataricus , Ss, using fluorescence polarization (FP) analysis, the dissociation constant (K _d ) As a result of measuring the value, it was shown that the K _d value was 756 nM for L-DNA binding (FIG. 3a).

In addition, using a gel-shift binding assay, five other species in the archaeal domain other than the archaea Sulfolobus solfataricus (Sulfolobus acidocaldarius [Sulfolobus acidocaldarius: Sa], Picrophilus torridus [ Picrophilus torridus: Pt], Metallosphaera sedula [Metallosphaera sedula: Ms], nitrosospira When Dpo4 was investigated from Vienensis [Nitrososphaera viennensis : Nv], Acidiplasma aeolicum : Aa), it was confirmed that all of them could bind to L-DNA (FIG. 6). _{Their K d} values for L-DNA binding were determined to be between 200 and 5088 nM. This suggests that the Dpo4 polymerase of the archaeal domain may generally have L-DNA binding properties, but the affinity may vary depending on the organism.

On the other hand, 5 other species in the archaea domain based on the S. solfataricus (Ss) Dpo4 (SEQ ID NO: 2) (Sa (SEQ ID NO: 11)], Pt (SEQ ID NO: 2) 12), Ms (SEQ ID NO: 13), As a result of confirming the homology of Dpo4 from Nv (SEQ ID NO: 14), Aa (SEQ ID NO: 15)) using the EMBOSS Needle program, 55.6% of Ss Dpo4 and Sa Dpo4 ( FIG. 8 ), Ss Dpo4 and Pt 48.2% for Dpo4 (Fig. 9), 56.8% for Ss Dpo4 and Ms Dpo4 (Fig. 10), 35.7% for Ss Dpo4 and Nv Dpo4 (Fig. 11), 44.5% for Ss Dpo4 and Aa Dpo4 (Fig. 12) ) was confirmed to show homology.

Through the above series of results, although there is a difference in L-DNA affinity depending on the derived organism, it has binding properties to L-DNA even if it shows only 30% homology with Dpo4 derived from Sulphorobus solfataricus. Could know.

Another aspect of the present invention provides an isolated L-DNA binding domain comprising the amino acid sequence represented by SEQ ID NO: 1.

In the present invention, the "L-DNA" is as described above.

As used herein, the term "L-DNA binding domain" or "isolated L-DNA binding domain" refers to a polypeptide that binds to L-DNA in vivo or in vitro. The 'polypeptide' is an amino acid polymer formed by peptide bonds of 10 or more amino acids, that is, the 'L-DNA binding domain' is a sequence of 121 amino acids (121 aa) represented by SEQ ID NO: 1. It means that it is composed of a polypeptide.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

The isolated L-DNA binding domain consisting of the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto, is at least 10%, 20%, 30%, 40%, 50%, 55%, 60% with SEQ ID NO: 1 %, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an L-DNA binding domain comprising an amino acid sequence that is at least 98%, 99%, or 100% identical.

In the present invention, the isolated L-DNA binding domain is derived from a DNA polymerase, the DNA polymerase is not limited thereto, but may be derived from DNA polymerase 4 (Dpo4).

The 'Dpo4 (DNA polymerase 4; DNA polyerase IV)' is a Y-family DNA polymerase derived from Sulfolobus solfataricus belonging to the thermophilic archaea. As a polymerase, it is one of the translesion synthesis (TLS) DNA polymerases. When Dpo4 encounters damaged DNA, it can proceed with DNA synthesis at the damaged site.

The Dpo4 is not limited thereto, but may be a polypeptide consisting of a sequence of 352 amino acids (352 a.a) represented by SEQ ID NO: 2.

In addition, the Dpo4 is, but not limited to, Sulfolobus solfataricus ( Sulfolobus solfataricus ), Sulfolobus acidocaldarius ( Sulfolobus acidocaldarius ), Picrophilus torridus ( Picrophilus torridus ), Metallos ferae three Doula ( Metallosphaera sedula ), nitrosospira Bienensis ( Nitrososphaera viennensis ), Acidiplasma aeolicum ( Acidiplasma aeolicum ) It may be derived from archaea.

In the present invention, the isolated L-DNA binding domain may be a LF domain (little finger domain) of DNA polymerase. Preferably, the LF domain of Dpo4, a Y-family DNA polymerase, may be a polypeptide consisting of the 121 amino acid (121 a.a) sequence shown in SEQ ID NO: 1.

On the other hand, Y-family polymerase consists of UmuC, DinB, Rev1/Rad30A genera, and Dpo4 has a DinB homolog. The DinB homology protein is a protein that exists widely in humans (DINB1), mice (DinB1), and in bacteria and archaea. DinB homologues contain the LF domain. The LF domain is composed of four β-sheets and two α-helixes βαββαβ.

In the present invention, the isolated L-DNA binding domain is the amino acid sequence shown in SEQ ID NO: 2 Arg (Arginine; R) at position 300, Lys at position 321 (Lysine; K) and Lys at position 339 (Lysine; K) One or more amino acid residues selected from the group consisting of may be unsubstituted or substituted (see FIG. 13 ).

In the present invention, the isolated L-DNA binding domain is Lys at position 300 (Lysine; K), Glu (Glutamic acid; E) at position 321 and Asn at position 339 (Asparagine; N) of the amino acid sequence shown in SEQ ID NO: 11 One or more amino acid residues selected from the group consisting of may not be substituted or mutated (see FIG. 13).

In the present invention, the isolated L-DNA binding domain is the amino acid sequence shown in SEQ ID NO: 12 Try (Tyrosine; Y), Lys at 321, Lys at position 321 (Lysine; K), and Lys at position 339 (Lysine; K) One or more amino acid residues selected from the group consisting of may be unsubstituted or substituted (see FIG. 13 ).

In the present invention, the isolated L-DNA binding domain is the amino acid sequence shown in SEQ ID NO: 13 Lys at 300 (Lysine; K), Lys at 321 (Lysine; K) and Lys at position 339 (Lysine; K) One or more amino acid residues selected from the group consisting of may be unsubstituted or substituted (see FIG. 13 ).

In the present invention, the isolated L-DNA binding domain is the amino acid sequence shown in SEQ ID NO: 14 Arg (Arginine; R) at position 300, Glu (Glutamic acid; E) at position 321, and Asp (Aspartic acid; D at position 339) ), one or more amino acid residues selected from the group consisting of may not be substituted or mutated (see FIG. 13 ).

In the present invention, the isolated L-DNA binding domain is the amino acid sequence shown in SEQ ID NO: 15 Try (Tyrosine; Y), 321 Arg (Arginine; R), and 339 Lys (Lysine; K) One or more amino acid residues selected from the group consisting of may be unsubstituted or substituted (see FIG. 13 ).

As used herein, the term “substitution” or “mutation” refers to alteration, deletion, or insertion of one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of the sequence.

The term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, eg, substitutions, insertions, or deletions.

The term “polynucleotide” refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical property. Double-stranded and single-stranded DNA and RNA are typical examples of polynucleotides.

The term “polypeptide” or “protein” refers to a molecule comprising at least two amino acid residues joined by peptide bonds to form a polypeptide. Small polypeptides of less than about 50 amino acids may be referred to as “peptides”.

In one embodiment of the present invention, it is confirmed that truncated Dpo4 (Dpo4-ΔLF) lacking the L-DNA binding domain, i.e., the LF domain, does not show significant binding affinity in the FP assay when tested for L-DNA binding. (Fig. 5a). In particular, it was confirmed that three amino acid residues in the LF domain are involved in binding to L-DNA ( FIGS. 5 b and 5 c ). Specifically, Arg300 and Lys339 of LF in chain A interact with phosphate near the major groove of the L-DNA duplex (Fig. 5b and 5c), and Arg300 and Lys321 of LF in chain B interact with the minor groove of the L-DNA duplex. It was confirmed that it interacts with phosphate (FIG. 5 b and 5 c). When this amino acid was mutated to alanine (Dpo4-mutLF), the K _d for L-DNA was 5520 nM, about a 7-fold decrease compared to the wild-type, demonstrating that these amino acids are key residues conferring L-DNA binding properties to Dpo4. (Fig. 5a).

That is, when (mutation) mutation of the 300Arg, 321Lys, 339Lys regions of the amino acid sequence of Dpo4, consisting of the amino acid sequence shown in SEQ ID NO: 2, specific binding between L-DNA and the LF domain of Dpo4 was lost. (FIGS. 5 and 13).

Another aspect of the present invention provides a protein comprising the domain.

In the present invention, the domain, specifically, the protein domain having binding affinity to L-DNA or the isolated L-DNA binding domain is as described above.

In the present invention, the protein including the domain may be applied to the preparation of antibody-drug conjugates (see FIG. 7 ).

The protein is a protein that exists in nature, and is not limited to a specific protein. Proteins are synthesized according to genetic information, and genes have information on the amino acid sequence of proteins. Protein is a polymer composed of 20 kinds of amino acids, and is a functional molecule in the living body, and is responsible for the processes and chemical reactions necessary for all kinds of life phenomena.

Proteins, for example, proteins that exist in nature, such as polymerase proteins, catalytic enzyme proteins, and hormone proteins, can be easily synthesized by those skilled in the art or isolated and obtained from living organisms in nature.

In the present invention, the protein is not particularly limited as long as the protein contains an L-DNA binding domain.

The 'L-DNA binding domain is included in the protein' means that the amino acid sequence information of the L-DNA binding domain, ie, SEQ ID NO: 1, is contained in the amino acid sequence information of a natural protein.

Although not limited thereto, the L-DNA binding domain included in the natural protein, that is, the LF domain derived from Dpo4 (DNA polymerase 4) is 10%, 20%, 30%, 40% with the amino acid sequence shown in SEQ ID NO: 1 , 50%, 55%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or may be one having 100% or more homology.

In the present invention, the term "antibody-drug conjugates" refers to an antibody that selectively attacks an antigen causing a disease in the human body and a drug having a therapeutic effect by a protein containing an L-DNA binding domain. It refers to a conjugated complex.

The 'protein including the L-DNA binding domain' is the same as described above, and the protein may be bound to a site other than the target recognition site of the antibody. Accordingly, as the antibody does not interfere with the target recognition site, that is, the antigen recognition site, a quantitative number of drugs (eg, one nucleic acid drug per antibody) can be labeled in a site-selective manner, so that a specific target selectively desired It is possible to deliver the drug by a quantity (see FIG. 7 ).

The term “antibody” refers to a protein comprising at least one immunoglobulin variable domain (variable region) or immunoglobulin variable domain (variable region) sequence. For example, an antibody may comprise a heavy chain variable region and a light chain variable region. Antibodies also include antigen-binding fragments of antibodies (eg, single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies) fragments (de Wildt et al , Eur J Immunol ., 26(3):629-39, 1996) as well as complete antibodies. Antibodies may have structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies can be derived from any source, but primates (human and non-human primates) and primatization are preferred.

In the present invention, the drug may be selected from the group consisting of an anticancer agent, an aptamer, an antisense oligonucleotide, a small interfering ribonucleic acid (siRNA, small interfering RNA), and a micro RNA (miRNA).

The drug is a protein or gene capable of hybridization with L-DNA binding to the L-DNA binding domain, that is, the LF domain (SEQ ID NO: 1) contained in the protein mediating the formation of the antibody-drug complex of the present invention. It may be a drug.

Another aspect of the present invention provides a drug delivery system comprising a protein comprising the L-DNA binding domain.

The 'protein comprising the L-DNA binding domain' is the same as described above.

As described above, the drug complementarily binds to the L-DNA backbone that binds to the L-DNA binding domain contained in the protein, that is, the LF domain (SEQ ID NO: 1), which mediates the formation of the antibody-drug complex of the present invention; Alternatively, it may be hybridized and delivered into cells (see FIG. 7 ).

Drugs that can be delivered into cells using the drug delivery system of the present invention, that is, the types of pharmaceutically active ingredients are not limited to specific ones, for example, anti-cancer agents, contrast agents, hormone agents, anti-hormones, vitamins, calcium agents, mineral agents, sugars agent, organic acid preparation, protein amino acid preparation, antidote, enzyme preparation, metabolic agent, diabetes combination agent, tissue revitalization agent, glophyll agent, pigment agent, tumor agent, tumor agent, radiopharmaceutical, tissue cell diagnostic agent, tissue cell therapy , antibiotic agent, antiviral agent, combination antibiotic agent, chemotherapeutic agent, vaccine, toxin, toxoid, antitoxin, leptospirin serum, blood product, biological agent, analgesic, immunogenic molecule, antihistamine, allergy drug, non-specific immunogen agent , anesthetics, stimulants, psychoactive agents, nucleic acids, aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNAs and microRNAs, and the like.

The drug delivery system of the present invention can be used to deliver a nucleic acid into a cell as a pharmaceutically active ingredient, and in this case, there is no concern that the sequence of the nucleic acid as the delivery material will be hybridized with the carrier sequence, so any natural nucleic acid-based delivery material can be freely used. It has the advantage of being able to use it.

Another aspect of the present invention is a drug delivery system comprising a protein comprising the L-DNA binding domain; And it provides a pharmaceutical composition for the treatment of cancer, including an anticancer agent.

The anticancer agent is not limited thereto, but may be a DNA aptamer or an RNA aptamer.

The pharmaceutical composition for the treatment of cancer of the present invention includes a pharmaceutically acceptable carrier together with the protein comprising the L-DNA binding domain described above. The pharmaceutically acceptable carrier is commonly used in formulation, and includes lactose. , dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995).

Meanwhile, the pharmaceutical composition for treating cancer according to the present invention may be administered by intravenous administration, intramuscular administration, subcutaneous administration, oral administration, intramedullary administration, transdermal administration, or topical administration, and may be administered as a solution, suspension injection, tablet or capsule. It can be formulated in various forms such as

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Preparation of Experimental Materials and Experimental Methods

Example 1-1: Preparation of experimental materials

All DNA polymerases except Dpo4 were purchased from New England Biolabs (USA). D-DNA oligonucleotides were purchased from Bioneer (Korea). L-DNA oligonucleotides were synthesized on a 1 μmol scale with a Mermaid-4 DNA/RNA synthesizer (Bioautomation, USA) using standard phosphoramidite chemistry. L-DNA phosphoamidite was purchased from ChemGenes (USA). Fluorescein phosphoamidite was purchased from Glen Research (USA). L-dTTP was purchased from TriLink BioTechnologies (USA). D-dNTPs were purchased from Promega (USA).

Example 1-2: Expression and purification of Dpo4

The pET21b plasmid carrying the codon-optimized S. solfataricus (Ss) Dpo4 was provided by the Yonsei University Synthetic Biology Laboratory. The recombinant plasmid was transformed into a C41 (DE3) expression host. Protein expression was induced with 1 mM isopropyl-β-D-thiogalactoside at 18° C. for 21 hours. Dpo4 (SEQ ID NO: 2) was purified using HisTrap and HiTrap monoS columns according to the procedure reported in Biochemistry, 43, 2106-2115 (2004). Purified protein was concentrated to 11 mg/ml in buffer 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT using a 10K out-off centrifugal filter.

Example 1-3: Gel mobility shift assay (GMSA)

Fluorescein in ThermoPol® buffer (20 mM Tris-HCl, 10 mM(NH ₄ ) ₂ SO ₄ , 10 mM KCl, 2 mM MgSO ₄ , 0.1% Triton X-100 pH 8.8@RT, New England Biolabs, USA) -labeled primer (GCT ACG ACT CAC TAT GGA CG, 100 nM) and template (AAA AAA AAA ACG TCC ATA GTG AGT CGT AGC, 100 nM) were heated to 95° C. for 5 minutes, annealed at room temperature for 10 minutes ( 25°C) to form a fluorescein-labeled DNA duplex. Polymerase (terminal deoxyribonucleotide transferase (TdT) (100 units)), Deep Vent (exo-) DNA polymerase (28 units), Dpo4 (10 μM), M-MuLV in DNA duplex solution Reverse transcriptase (2800 units), Klenow fragment (3'→5' exo-) (70 units), Taq DNA polymerase (70 units), and Vent (exo-) DNA polymerase (28 units)) were added and , the mixture was incubated for 30 min at room temperature. The mixture was then analyzed in a 6% non-denaturing PAGE run in 0.5×TBE (44.5 mM Tris-HCl, 44.5 mM boric acid, 1 mM EDTA pH 8.0) at 100V. Bands were visualized using Chemidoc (Biorad, USA).

Example 1-4: Fluorescence Polarization Analysis

Fluorescein-labeled DNA duplex (100 nM) consisting of fluorescein-labeled primer (fluorescein-GGG GGA AGG ATT CC) and template (TCA CGG AAT CCT TCC CCC) was mixed with Dpo4 reaction buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM MgCl ₂ , 5 mM DTT, 10% glycerol, 0.1 mM EDTA) at various concentrations (0-10 μM) of Dpo4 and incubated at 4° C. for 30 minutes. The binding affinity of Dpo4 to DNA duplexes was analyzed by fluorescence polarization values and measured using Appliskan (Thermo Fisher Scientific, USA). K _d values were estimated based on non-linear regression to the one-site saturated ligand binding equation using Sigmaplot software (Systat, USA). For competition experiments, various concentrations (0-4 µM) of D-DNA duplexes were added to solutions of Dpo4 (1 µM) pre-mixed with fluorescein-labeled L-DNA duplexes (100 nM) in Dpo4 reaction buffer. was added and incubated at room temperature for 30 minutes. Fluorescence polarization values were measured using Appliskan (Thermo Fisher Scientific, USA). IC ₅₀ values were estimated based on nonlinear regression to a four parameter logistic curve equation using Sigmaplot software (Systat, USA). All data were obtained from triplicate independent experiments.

Example 1-5: Primer Extension

Prior to extension analysis, 5'-fluorescein-labeled DNA primer (fluorescein-GCT ACG AGC ACT ATG GA CG) and DNA template (AAA AAA AAA ACG TCC ATA GTG AGT CGT AG C) were incubated at 95° C. for 2 min. It was annealed by heating with a furnace and slowly cooling to 4°C. Primer extension reaction was 20 mM Tris-HCl (pH 8.8 ), 10 mM (NH 4) 2 SO- 4, 10 mM KCl, 2 mM MgSO 4, 0.1% Triton X-100, 1 mM dTTP and Dpo4 (1 μM) was performed using 1 μM of primer/template in a buffer containing The reaction was carried out at 55° C. for 30 minutes and terminated by addition of equal volumes of 95% formamide and 10 mM EDTA followed by incubation at 95° C. for 5 minutes. Reaction products were digested on PAGE 20% denaturing in 7M urea and visualized with an iBright FL1000 (Invitrogen).

Example 1-6: Isothermal titration calorimetry

Isothermal titration calorimetry of Dpo4 and DNA was measured on a Nano ITC (TA Instruments, USA). Dpo4 and DNA were dialyzed against ITC buffer at pH 7.5 containing 20 mM HEPES, 100 mM NaCl, 0.5 mM EDTA, 0.5 mM TCEP, and 10 mM MgSO _{4 .} The optimized Dpo4 concentration was 25 μM and the DNA concentration was 125 μM. 5 μL DNA from the sample cells was automatically titrated with Dpo4 solution, and a total of 20 titrations were performed with 300 sec intervals between each shot to reach complete equilibrium. The total measurement time in individual experiments was over 6,000 seconds. ITC of Dpo4 to L-DNA was performed at 45°C, and ITC of Dpo4 to D-DNA was performed at 15°C. Data were analyzed with the appropriate program Nanoanalyze (TA Instruments, USA) for binding models where the binding rate is independent.

Examples 1-7: Crystallization and Structure Determination

DPO4 and DNA duplex formed from primer (GGG GGA AGG ATT CC) and template (TCA CGG AAT CCT TCC CCC) were incubated at about 1:1.3 molar ratio at 4°C. Determination of Dpo4/D-DNA or Dpo4/L-DNA complexes was prepared using a 5:5 proportional volume of protein/DNA solution and a reservoir solution (0.1 M BIS-TRIS (pH 7.0), 0.1 M calcium acetate, 2%, respectively). glycerol, and 9% w/v PEG3350) or reservoir solution (0.1 M sodium formate, 0.05 M BICINE (pH 8.5), 10% w/v polyethylene glycol monomethyl ether 5,000, and 1.1% benzamidine-HCl ) was grown using a sitting-drop vapor diffusion method in a drop containing at 19 °C. 0.1 M BIS-TRIS (pH 7.0), 20% PEG3350, 100 mM CaOAC2, and 25% glycerol for Dpo4/D-DNA crystals, and 0.1 M sodium formate, 0.05 M BICINE (pH) for Dpo4/L-DNA crystals 8.5), 10% w/v polyethylene glycol monomethyl ether 5,000, 1.1% benzamidine-HCl and 25% glycerol were rapidly immersed in the crystals, which were immediately flash frozen with liquid nitrogen. Data were collected at a wavelength of 1.0 Å at the Pohang accelerator light source beamline 5C (Pohang, Korea). The data set was reduced and enlarged using HKL-2000 ( Method Enzymol , 276:307-326, 1997). Dpo4/D-DNA and Dpo4/L-DNA crystals were crystallized at P22 ₁ 2 ₁ and P12 ₁ 1 , and diffracted at resolutions of 2.60 and 2.36 Å, which were found to be 2.60 and 2.36, respectively. Phases for both complexes were determined through molecular substitution using the published DPO4 structure (PDB 2BQ3) ( J Biol Chem , 280:29750-29764, 2005), and initial model construction was based on PHENIX ( Acta Crystallogr D , 66:213-221, 2010) using Autobuild. Model building and refinement were performed using COOT ( Acta Crystallogr D , 60:2126-2132, 2004) and PHENIX. The final model was validated through MOLPROBITY ( Acta Crystallogr D , 66:12-21, 2010). Statistics are summarized in Table 2.

Example 1-8: Acquisition of accession number of crystal structure

Crystal structures were deposited in the RCSB Protein Data Bank with accession code 6L84 for DPO4/D-DNA duplex and accession code 6L97 for DPO4/L-DNA duplex.

Examples 1-9: Alignment of Dpo4 from various organisms

5 different species (Sulpholobus acidocaldarius) in the archaea domain based on S. solfataricus : Ss) Dpo4 (SEQ ID NO: 2) obtained in Example 1-2 [ Sulfolobus acidocaldarius : Sa (SEQ ID NO: 11)], Picrophilus torridus [Picrophilus torridus: Pt (SEQ ID NO: 12)], Metallosphaera sedula [Metallosphaera sedula: Ms (SEQ ID NO: 13)], nitrosospira Dpo4 (see Table 3) from Vienensis [Nitrososphaera viennensis : Nv (SEQ ID NO: 14)], Acidiplasma aeolicum : Aa (SEQ ID NO: 15)) was transferred to the EMBOSS Needle (https:// Using www.ebi.ac.uk/Tools/psa/emboss_needle/), pairwise sequence alignment was performed to confirm homology.

As a result, in the case of Ss Dpo4 and Sa Dpo4 55.6% (FIG. 8), in the case of Ss Dpo4 and Pt Dpo4 48.2% (FIG. 9), in the case of Ss Dpo4 and Ms Dpo4 56.8% (FIG. 10), Ss Dpo4 and Nv Dpo4 of 35.7% (Fig. 11), and 44.5% (Fig. 12) of Ss Dpo4 and Aa Dpo4 (Fig. 12).

Example 2: Analysis of Binding of DNA Polymerase to D-DNA and L-DNA

DNA polymerase is a protein enzyme that extends primer DNA by binding deoxynucleoside triphosphate (dNTP) to the 3'-end of the primer. The polymerase binds to the D-DNA duplex in a chiral-selective manner using key domains for enzymatic activity, such as thumb, palm and finger ( FIG. 1 ). Although the chiral selectivity of polymerases for DNA binding is believed to be very faithful, it is not known whether there are in fact any polymerases with L-DNA binding properties. In this regard, the present inventors screened a series of readily available DNA polymerases to investigate whether the polymerase could bind to the L-DNA duplex. Surprisingly, we found that Dpo4, M-MuLV reverse transcriptase (RT) and Klenow fragment (KF) ( FIG. 2 ) can bind to the L-DNA duplex. In the present invention, the L-DNA binding properties of the smallest polymerase, Dpo4, were specifically investigated. After determining the dissociation constant for DNA binding, we also obtained crystal structures of polymerase complexed with D-DNA and L-DNA, and characterized the amino acid residues and domains responsible for DNA binding with their respective chirality. The structure exhibited markedly different binding behavior, complex morphology, and binding stoichiometry of Dpo4 to L-DNA compared to D-DNA in the major binding domain. This is the first study to report the intrinsic L-DNA duplex-binding properties of native DNA polymerases.

Example 3: Analysis of binding capacity of DNA polymerase 4 (Dpo 4) to D-DNA and L-DNA

Dpo4 from Sulfolobus solfataricus (Ss) was bacterially expressed and purified according to a previous study (KA Fiala & Z. Suo, Biochemistry , 43:2106-2115, 2004). To investigate the DNA binding ability of Dpo4, the dissociation constant (K _d ) value was measured using fluorescence polarization (FP) analysis, which indicated that the K _d value was 187 nM for D-DNA binding and L-DNA binding. to 756 nM (FIG. 3a). _{The K d} values for Dpo4/D-DNA complexes estimated by FP analysis were higher than previously reported values (36 nM), which may be due to different assay conditions and methods (KA Fiala et.al. , Journal of Biological Chemistry , 282:8188-8198, 2007). To confirm that L-DNA binding can perturb and replace D-DNA bound to Dpo4, we also performed a competition analysis for DNA-Dpo4 interaction using the FP method (Fig. 3b). . The high FP level of fluorescently labeled L-DNA complexed with polymerase was reduced upon addition of unlabeled D-DNA, indicating that D-DNA was combined with L-DNA (IC ₅₀ : 670 nM) for binding to polymerase. indicates competition. This suggests that Dpo4 has covalent binding sites for D-DNA and L-DNA. Regarding the polymerization activity, Dpo4 was unable to bind any nucleotides to the 3'-end of the L-DNA primer, but could add both D-dTTP and L-dTTP to the 3'-end of the D-DNA primer. There was (Fig. 3c). This suggests that the binding mode of L-DNA in Dpo4 is not the same as that of D-DNA, and there is a covalent binding site, but it is not suitable for polymerase activity. When Dpo4/L-DNA complexes were tested using additional L-DNA duplexes with different sequences (Table 1), the K _d values varied with the sequence, but Dpo4 bound to all tested L-DNA duplexes. Could. This demonstrates that the L-DNA binding properties of Dpo4 are not highly dependent on the DNA sequence (Fig. 3d).

D-DNA and L-DNA sequences tested for Dpo4 binding designation order SEQ ID NO: GC-0-L 5'-AAAAAAAAAAAAAAAAAA-FAM-3' 3 GC-0-D 3'-TTTTTTTTTTTTTTTTTT-5' 4 GC-33-L 5'-CAATGTCTAAACTGAAAG-FAM-3' 5 GC-33-D 3'-GTTACAGATTTGACTTTC-5' 6 GC-67-L 5'-GGACCGTGACTCCCTGAC-FAM-3' 7 GC-67-D 3'-CCTGGCACTGAGGGACTG-5' 8 GC-100-L 5'-GGGGGGGGGGGGGGGGGG-FAM-3' 9 GC-100-D 3'-CCCCCCCCCCCCCCCCCC-5' 10

* Indicates FAM fluorescein.

Example 4: Analysis of the crystal structure of Dpo4 complexed with D-DNA and L-DNA

After revealing that Dpo4 has an affinity for L-DNA as well as D-DNA, the present inventors investigated the crystal structure of the polymerase complex with each DNA stereoisomer in order to elucidate the molecular aspect of the protein-DNA interaction. The present inventors obtained crystals of Dpo4/D-DNA and Dpo4/L-DNA complexes belonging to the _{space groups P22 1} 2 ₁ and P12 _{1 1 and diffracted to 2.6 and 2.36 Å, respectively (Table 2).} Both structures were found through molecular replacement using the previously reported Dpo4 structure (PDB 2BQ3) (H. Zang et.al., Journal of Biological Chemistry , 280:29750-29764, 2005).

Data collection and improvement statistics Dpo4 D-DNA L-DNA data collection space group P 2 2 ₁ 2 ₁ P 1 2 ₁ 1 unit cells (a, b, and c; Å)
(α, β, and γ; °) 52.8, 98.7, 100.8
90.0, 90.0, 90.0 85.9, 55.0, 96.6
90.0, 113.4, 90.0 Wavelength (Å) 1.0 0.9794 Resolution ^a (Å) 50-2.60 (2.64-2.60) 50-2.36 (2.44-2.36) R _merge ^a,b (%) 12.2 (35.9) 12.5 (55.5) mean I/σ ( I ) ^a 20.6 (5.4) 28.5 (3.1) Completeness ^a (%) 99.8 (99.8) 97.9 (95.3) redundancy ^a 11.3 (7.7) 4.6 (3.0) Observed reflections (unique) 188,903 (16,695) 153,932 (33,537) Wilson B-factor 35.1 50.5 refinement R _work /R _free ^c (%) 18.2/22.6 23.0/26.6 asu (air separation unit) glycoprotein glycoprotein residues 341 672 water molecules per asu 108 115 Other ligands per asu 29 28 CA 3 Ramachandran plot Preferred/acceptable/outlier (%) 95.6/4.1/0.3 95.3/4.7/0.0 root mean square (Rms) deviation bond length (Å) 0.0034 0.70 bonding angle (°) 0.475 2.09 mean B factor (Å ² ) 42.7 66.8 macromolecules 42.8 66.2 ligand 52.8 74.4 menstruum 40.68 55.9 PDB code 6L84 6L97

^a Numbers in parentheses indicate outer-resolution shells.

^b R _merge =[Σ _hkl Σ _i | I -< I >|/Σ _hkl Σ _I | I |×100].

^c R _factor / R _free =Σ _hkl │ F _o |-| F _c │/Σ _hkl | F _o |, where F _o and F _c are the observed and calculated structure factors, respectively.

The structure of the Dpo4/D-DNA complex was similar to that of the known Dpo4/D-DNA binary complex (H. Ling et.al. , Cell , 2001, 107:91-102). The D-DNA duplex is embedded in the four domains of Dpo4 (thumb, palm, finger, and little finger) as if the right hand is holding the 'rope', which is a Y-family DNA polymerase complexed with double-stranded DNA. (Fig. 4a) [V. Jha & H. Ling, Sci Rep , 8:15125-15125, 2018; W. Yang, Biochemistry , 53:2793-2803, 2014; Y. Zhao et.al. , Proceedings of the National Academy of Sciences , 109:7269-7274, 2012]. The thumb domain interacted with the minor groove of the D-DNA duplex. An active site residue having a divalent ion (Ca ²⁺ , yellow sphere) critical for polymerization activity was located in the palm domain. A finger domain having a role of interacting with incoming nucleotides was positioned at the 3'-end of the primer to prepare elongation. Little finger domains are known to improve the processivity of Dpo4 in the polymerization of D-DNA (H. Ling et.al. , Cell , 107:91-102, 2001). To provide a binding interface between the polymerase and the major groove of the D-DNA duplex, and to extrude the 5'-end of the template strand, a narrow gap was made with the finger domain (Fig. 4a).

In stark contrast to the structure of the Dpo4/D-DNA complex, which showed a typical DNA-polymerase interaction, the structure of the Dpo4/L-DNA complex exhibited a non-canonical DNA-polymerase binding interface. The L-DNA duplex did not have significant interactions with the three major domains of the polymerase (thumb domain, palm domain, and finger domain), but from two Dpo4 molecules forming a dimer (chain A and chain B). It took a 'pinky swear' structure made by two little finger domains (FIG. 4 b). As shown in Fig. 4c, the overall structure of this dramatically modified polymerase was mainly due to the location of the little finger away from other domains (Fig. 4c, top). apo-Dpo4 (PDB: 2RDI) [JH Wong et.al. , Journal of Molecular Biology , 379:317-330, 2008] and making a hinge around residues 237-240 to bring the little finger domain closer to the finger domain in the Dpo4/D-DNA complex. The linker (residues 231-243) was relaxed to interact with L-DNA located outside the active pocket. However, regardless of the presence and chirality of DNA, the folding of each domain was conserved as revealed by aligning each domain from the Dpo4/D-DNA and Dpo4/L-DNA complexes (root mean square deviation (RMSD)). : 0.590 for thumb, 0.302 for palm, 0.480 for finger, and 0.298 for little finger (Fig. 4c, bottom). The little finger domain of Dpo4 dimerized in complex with L-DNA forms a β-barrel-like structure, providing a binding interface to L-DNA (Fig. 4d). Thus, the position of the little finger domain also determines the binding stoichiometry of the polymerase to DNA, such as 1:1 with D-DNA and 2:1 with L-DNA. When the present inventors measured the binding ratio between Dpo4 and DNA using isothermal titration calorimetry (ITC), Dpo4 was bound to D-DNA in a ratio of 1:0.9 whereas 1:0.5 (or 2:1) ) was confirmed to bind to L-DNA at a ratio of ( FIG. 4 e ). That is, it was confirmed that 2 molecules of the protein and 1 molecule of the L-DNA duplex were bound on the crystal structure, meaning that they were bound in a ratio of 2:1 even in the actual solution.

Example 5: Analysis of specific interactions between L-DNA and the LF (little finger) domain of Dpo4

According to the structure of the Dpo4/L-DNA complex, L-DNA only interacted with the dimerized little finger domain of Dpo4, indicating that little finger is important for possessing L-DNA binding properties to Dpo4. Indeed, it was confirmed that the truncated Dpo4 lacking the little finger domain (Dpo4-ΔLF) did not show significant binding affinity in the FP assay when tested for L-DNA binding (Fig. 5a). However, the little finger to keep the L-DNA binding properties with only domain, full-length (full length) similar K _d values and Dpo4 showed the (K _d values of 860 nM) (in Fig. 5 a), this revealed by the crystal structure Little finger-biased binding modes can be supported consistently. In particular, three amino acid residues in the two finger domains of the Dpo4 dimer were involved in binding to L-DNA ( FIGS. 5 b and 5 c ). The binding interface may be based on electrostatic interactions between the positively charged side chains of these residues and the negatively charged phosphate backbone of L-DNA. In chain A, Arg300 and Lys339 of the little finger interacted with phosphate near the major groove of the L-DNA duplex (Fig. 5b and Fig. 5c), and Arg300 and Lys321 of the little finger in chain B interacted with the phosphate of the minor groove of the L-DNA duplex. interacted (FIG. 5B and 5C). When this amino acid was mutated to alanine (Dpo4-mutLF), the K _d for L-DNA was 5520 nM, about a 7-fold decrease compared to the wild-type, demonstrating that these amino acids are key residues conferring L-DNA binding properties to Dpo4. (Fig. 5a). Taken together, these results show that the little finger domain (LF domain; SEQ ID NO: 1) of Dpo4 plays an important role in L-DNA recognition.

Example 6: Analysis of L-DNA binding capacity of Dpo4 from various organisms

DNA and proteins with mirror image chirality are expected to interact with each other in a mirrored manner, but identical to native DNA and proteins. Previously, it was reported that synthetic mirror HIV-1 protease has protease activity only on the mirror peptide substrate (R. Milton, S. Milton & S. Kent, Science , 256:1445-1448, 1992). More recently, synthetic mirror DNA polymerases were synthesized chemically and exhibited polymerization activity on mirror DNA to generate mirror DNA replicons (A. Pech et. al. , Nucleic Acids Research , 45:3997- 4005, 2017; Z. Wang et. al. , Nature Chemistry , 8:698, 2016). Naturally developed chirality preferences, such as D-DNA for L-proteins, remain stringent in DNA-protein interactions, but mirror-natural interactions (L-DNA/RNAs for L-proteins) or cross-chirality interactions DNA and RNA showing action can still be artificially developed using SELEX (A. Vater & S. Klussmann, Drug Discovery Today , 20:147-155, 2015). Ribozymes with cross-chiral polymerization activity have also been developed (JT Sczepanski & GF Joyce, Nature , 515:440, 2014). In contrast to these studies, the present invention describes the inherently present cross-chiral L-DNA binding potential of Dpo4. To the best of our knowledge, this is the first discovery of a cross-chiral interaction of a native protein with mirror DNA in a sequence-independent manner.

The importance of the little finger domain for polymerases to recognize L-DNA suggests that Dpo4 polymerases with little fingers from other organisms may also have L-DNA binding properties. Indeed, the present inventors used a gel-shift binding assay to study five different species in the archaea domain ( Sulfolobus acidocaldarius [Sa], Pycrophilus). Torridus [ Picrophilus torridus: Pt], Metallosphaera sedula [ Metallosphaera sedula: Ms], nitrosospira When Dpo4 (see Table 3) was investigated from Vienensis [Nitrososphaera viennensis : Nv], Acidiplasma aeolicum : Aa), it was confirmed that they were all capable of binding to L-DNA ( Fig. 6). _{Their K d} values for L-DNA binding were determined to be between 200 and 5088 nM. This indicates that the Dpo4 polymerase of the archaeal domain may generally have L-DNA binding properties, but the affinity may vary depending on the organism.

Dpo4 polymerase amino acid sequence of 5 archaeal species archaea species order SEQ ID NO: Sulfolovus Acidocaldarius
( Sulfolobus acidocaldarius : Sa) MIVIFVDFDYFFAQVEEVLNPQYKGKPLVVCVYSGRTKTSGAVATANYEARKLGVKAGMPIIKAMEIAPNAVYLPMRKPVYEAFSNRIMNLLNKYADKIEIASIDEAYLDVTNKVQGNFELGVELARKIKQEILEKEKITVTVGVTPNKILAKIIADKSKPNGLGVIRPTEVQDFLNELDIDEIPGIGSVLARRLNELGIHKLRDILSKNYNELEKITGKAKALYLLKLAENKYSEPVENKSKIPHGRYLTLPYNTRDVKVILPYLKKAINEAYNKVNGIPMRITVVAIMEDLDILSKGKKFKHGISIDNAYKVAEDLLRELLIRDKRRNIRRIGVKLDNIIINKTNLSDFFDI 11 picrophilus toridus
( Picrophilus torridus: Pt) MIMLIDFDYFFAQVEEINDPSLKGKPVVVSVYSGRNERSGAVATSNYEARALGIKSGMPLYRALEIGKNRAVFLPIRKDFYQKYSDKIMDIISEYSEKMEIASIDEAYIDIDGNDCKIGIANEIKNRILNETGIKVSIGIGINKVIAKMAAEMAKPNGIKCISADETGEFLNNIKINDIPGIGKVLSKNLNEIGIEYLRDIKNFDVNKIKSILGESKTNYLYELYENKYFSPVEPRVKKNFGRYLTLPENTRDIDKIVPYLKKSIDAAYEKAPGIPQEISVVAIMEDLDIVSRSYTGNAIKRDDSINIALNLLNKIISEDNRNIRRIGVRLSKISKNNTLDDFF 12 Metalos Spera Sedula
( Metallosphaera sedula : Ms.) MIVLFVDFDYFFAQVEEILNPSLKGKPVVVCVYSGRTKDSGAVATSNYEARKLGIKAGMPIIKAKEIGKDAVFLPMRKEVYQQVSRRVMNIISGYGDKLEIASIDEAYLDITRRVKDFDEAKELARKLKAEVLEKERLRVTVGIGPNKVVAKIIADMNKPDGLGIIYPEEVKDFLYNLDISKVPGVGKITEEILRKVGINRLGDVINKSGELVNLVGKSKANYLLSLANNTYHDPVESREITHRGRYVTLPENTRDLNRILPSLKRSIEEAYSKVDGIPMEIYVVAIMEDLDIVSKGKSFKFGVSQDRALSVAQELLNKILESDKRKLRRVGVRLGKITKSSTLEDFLH 13 nitrosospira vienensis
( Nitrososphaera viennensis : Nv) MLAPPHRVVMHIDFDYFFAQCEEVRRPEIRQRPVLVCVFSGRTEDSGVVSTANYVARKYGVKSGIPIRVAKAKLAGVDDALFLPLDASYYRQVSENAMSAIKEHADVFEHVGIDECFIDVSGRANGRFDAAEALARTVKQRVQERTKLTCSVGVAPNKMLAKIASDYNKPDGLTVVRPENALQFVSALDVDKIPGIGPKTRDRLAELGVKTAGDLASFDLFRLIKEFGKKTATYIHNAAKGIDDEPVMESAEGERRQQIMRIVTLKKDAQSAEEMYADLEEICRDVYESATEKKVAFGSVGIILVLDDLENVTRSRGLKAHASSFELLHSTARSLLDEAMGEKKRSVRRLGVRISDFQDSSGQNTLFDYFTARQGE 14 acidiplasma aericum
( Acidiplasma aeolicum : Aa) MPQFIIFIDFDYFFAQVEEILNPEIREKPVVVCVYSGRTETSGAVATSNYLARKIGIKSGMPLPQAMKIGKDRAVFLPIRKDIYKEWSDHIMDIISEYSDKIEIASIDEAYIDVTDSCKDFNDAVNTAQKIKDEIYNKTGVRVSVGVSVNKAIAKVLGDMAKPNGIKSVGPSEIQDFLKGLEISKIPGVGKVIGQRLNDAGIKYITDVINADRDQLINIIGTAKYNYLYDIANNTYNKPVTPREKKNFGRYMTLPENTRDVNIIIPYVKKAIDSAYEKAPGLPSELSLVAIMEDISILSRSYTGARIDKNRALEIAIGLLNRIINEDQRNIRRVGVRLGKITKNETLDDFF 15

In conclusion, the present inventors revealed the L-DNA duplex binding properties of Dpo4. According to the structure revealed by crystallography, Dpo4 dimerized to establish a little finger-based binding interface for interacting with L-DNA with a 2:1 binding ratio, but the activity formed by all domains with a 1:1 binding ratio. A D-DNA duplex was embedded in the pocket. The little finger domain was important for Dpo4 to have L-DNA binding properties, as the affinity for L-DNA was reduced or lost when the little finger domain was mutated or truncated, respectively. Our study characterizing the molecular aspects of the L-DNA binding properties of natural polymerases will contribute to understanding the principles of cross-chirality interactions between L-DNA duplexes and never-explored native proteins.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> An L-DNA-binding natural protein and uses thereof <130> DP-2019-0467 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> LF domain <400> 1 Tyr Asn Glu Pro Ile Arg Thr Arg Val Arg Lys Ser Ile Gly Arg Ile 1 5 10 15 Val Thr Met Lys Arg Asn Ser Arg Asn Leu Glu Glu Ile Lys Pro Tyr 20 25 30 Leu Phe Arg Ala Ile Glu Glu Ser Tyr Tyr Lys Leu Asp Lys Arg Ile 35 40 45 Pro Lys Ala Ile His Val Val Ala Val Thr Glu Asp Leu Asp Ile Val 50 55 60 Ser Arg Gly Arg Thr Phe Pro His Gly Ile Ser Lys Glu Thr Ala Tyr 65 70 75 80 Ser Glu Ser Val Lys Leu Leu Gln Lys Ile Leu Glu Glu Asp Glu Arg 85 90 95 Lys Ile Arg Arg Ile Gly Val Arg Phe Ser Lys Phe Ile Glu Ala Ile 100 105 110 Gly Leu Asp Lys Phe Phe Asp Thr 115 120 <210> 2 <211> 352 <212> PRT <213> Artificial Sequence <220> <223> Ss Dpo4 <400> 2 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GC-0-L <400> 3 aaaaaaaaaa aaaaaaaa 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GC-0-D <400> 4 tttttttttt tttttttt 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GC-33-L <400> 5 caatgtctaa actgaaag 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GC-33-D <400> 6 gttacagatt tgactttc 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GC-67-L <400> 7 ggaccgtgac tccctgac 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GC-67-D <400> 8 cctggcactg agggactg 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GC-100-L <400> 9 gggggggggg gggggggg 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GC-100-D <400> 10 cccccccccc cccccccc 18 <210> 11 <211> 354 <212> PRT <213> Artificial Sequence <220> <223> Sa Dpo4 <400> 11 Met Ile Val Ile Phe Val Asp Phe Asp Tyr Phe Phe Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Gln Tyr Lys Gly Lys Pro Leu Val Val Cys Val 20 25 30 Tyr Ser Gly Arg Thr Lys Thr Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Leu Gly Val Lys Ala Gly Met Pro Ile Ile Lys Ala 50 55 60 Met Glu Ile Ala Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Pro Val 65 70 75 80 Tyr Glu Ala Phe Ser Asn Arg Ile Met Asn Leu Leu Asn Lys Tyr Ala 85 90 95 Asp Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Val Thr 100 105 110 Asn Lys Val Gln Gly Asn Phe Glu Leu Gly Val Glu Leu Ala Arg Lys 115 120 125 Ile Lys Gln Glu Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly 130 135 140 Val Thr Pro Asn Lys Ile Leu Ala Lys Ile Ile Ala Asp Lys Ser Lys 145 150 155 160 Pro Asn Gly Leu Gly Val Ile Arg Pro Thr Glu Val Gln Asp Phe Leu 165 170 175 Asn Glu Leu Asp Ile Asp Glu Ile Pro Gly Ile Gly Ser Val Leu Ala 180 185 190 Arg Arg Leu Asn Glu Leu Gly Ile His Lys Leu Arg Asp Ile Leu Ser 195 200 205 Lys Asn Tyr Asn Glu Leu Glu Lys Ile Thr Gly Lys Ala Lys Ala Leu 210 215 220 Tyr Leu Leu Lys Leu Ala Glu Asn Lys Tyr Ser Glu Pro Val Glu Asn 225 230 235 240 Lys Ser Lys Ile Pro His Gly Arg Tyr Leu Thr Leu Pro Tyr Asn Thr 245 250 255 Arg Asp Val Lys Val Ile Leu Pro Tyr Leu Lys Lys Ala Ile Asn Glu 260 265 270 Ala Tyr Asn Lys Val Asn Gly Ile Pro Met Arg Ile Thr Val Val Ala 275 280 285 Ile Met Glu Asp Leu Asp Ile Leu Ser Lys Gly Lys Lys Phe Lys His 290 295 300 Gly Ile Ser Ile Asp Asn Ala Tyr Lys Val Ala Glu Asp Leu Leu Arg 305 310 315 320 Glu Leu Leu Ile Arg Asp Lys Arg Arg Asn Ile Arg Arg Ile Gly Val 325 330 335 Lys Leu Asp Asn Ile Ile Ile Asn Lys Thr Asn Leu Ser Asp Phe Phe 340 345 350 Asp Ile <210> 12 <211> 344 <212> PRT <213> Artificial Sequence <220> <223> Pt Dpo4 <400> 12 Met Ile Met Leu Ile Asp Phe Asp Tyr Phe Phe Ala Gln Val Glu Glu 1 5 10 15 Ile Asn Asp Pro Ser Leu Lys Gly Lys Pro Val Val Val Ser Val Tyr 20 25 30 Ser Gly Arg Asn Glu Arg Ser Gly Ala Val Ala Thr Ser Asn Tyr Glu 35 40 45 Ala Arg Ala Leu Gly Ile Lys Ser Gly Met Pro Leu Tyr Arg Ala Leu 50 55 60 Glu Ile Gly Lys Asn Arg Ala Val Phe Leu Pro Ile Arg Lys Asp Phe 65 70 75 80 Tyr Gln Lys Tyr Ser Asp Lys Ile Met Asp Ile Ile Ser Glu Tyr Ser 85 90 95 Glu Lys Met Glu Ile Ala Ser Ile Asp Glu Ala Tyr Ile Asp Ile Asp 100 105 110 Gly Asn Asp Cys Lys Ile Gly Ile Ala Asn Glu Ile Lys Asn Arg Ile 115 120 125 Leu Asn Glu Thr Gly Ile Lys Val Ser Ile Gly Ile Gly Ile Asn Lys 130 135 140 Val Ile Ala Lys Met Ala Ala Glu Met Ala Lys Pro Asn Gly Ile Lys 145 150 155 160 Cys Ile Ser Ala Asp Glu Thr Gly Glu Phe Leu Asn Asn Ile Lys Ile 165 170 175 Asn Asp Ile Pro Gly Ile Gly Lys Val Leu Ser Lys Asn Leu Asn Glu 180 185 190 Ile Gly Ile Glu Tyr Leu Arg Asp Ile Lys Asn Phe Asp Val Asn Lys 195 200 205 Ile Lys Ser Ile Leu Gly Glu Ser Lys Thr Asn Tyr Leu Tyr Glu Leu 210 215 220 Tyr Glu Asn Lys Tyr Phe Ser Pro Val Glu Pro Arg Val Lys Lys Asn 225 230 235 240 Phe Gly Arg Tyr Leu Thr Leu Pro Glu Asn Thr Arg Asp Ile Asp Lys 245 250 255 Ile Val Pro Tyr Leu Lys Lys Ser Ile Asp Ala Ala Tyr Glu Lys Ala 260 265 270 Pro Gly Ile Pro Gln Glu Ile Ser Val Val Ala Ile Met Glu Asp Leu 275 280 285 Asp Ile Val Ser Arg Ser Tyr Thr Gly Asn Ala Ile Lys Arg Asp Asp 290 295 300 Ser Ile Asn Ile Ala Leu Asn Leu Leu Asn Lys Ile Ile Ser Glu Asp 305 310 315 320 Asn Arg Asn Ile Arg Arg Ile Gly Val Arg Leu Ser Lys Ile Ser Lys 325 330 335 Asn Asn Thr Leu Asp Asp Phe Phe 340 <210> 13 <211> 349 <212> PRT <213> Artificial Sequence <220> <223> Ms Dpo4 <400> 13 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Phe Ala Gln Val Glu 1 5 10 15 Glu Ile Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Tyr Ser Gly Arg Thr Lys Asp Ser Gly Ala Val Ala Thr Ser Asn Tyr 35 40 45 Glu Ala Arg Lys Leu Gly Ile Lys Ala Gly Met Pro Ile Ile Lys Ala 50 55 60 Lys Glu Ile Gly Lys Asp Ala Val Phe Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Arg Arg Val Met Asn Ile Ile Ser Gly Tyr Gly 85 90 95 Asp Lys Leu Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Thr 100 105 110 Arg Arg Val Lys Asp Phe Asp Glu Ala Lys Glu Leu Ala Arg Lys Leu 115 120 125 Lys Ala Glu Val Leu Glu Lys Glu Arg Leu Arg Val Thr Val Gly Ile 130 135 140 Gly Pro Asn Lys Val Val Ala Lys Ile Ile Ala Asp Met Asn Lys Pro 145 150 155 160 Asp Gly Leu Gly Ile Ile Tyr Pro Glu Glu Val Lys Asp Phe Leu Tyr 165 170 175 Asn Leu Asp Ile Ser Lys Val Pro Gly Val Gly Lys Ile Thr Glu Glu 180 185 190 Ile Leu Arg Lys Val Gly Ile Asn Arg Leu Gly Asp Val Ile Asn Lys 195 200 205 Ser Gly Glu Leu Val Asn Leu Val Gly Lys Ser Lys Ala Asn Tyr Leu 210 215 220 Leu Ser Leu Ala Asn Asn Thr Tyr His Asp Pro Val Glu Ser Arg Glu 225 230 235 240 Ile Thr His Arg Gly Arg Tyr Val Thr Leu Pro Glu Asn Thr Arg Asp 245 250 255 Leu Asn Arg Ile Leu Pro Ser Leu Lys Arg Ser Ile Glu Glu Ala Tyr 260 265 270 Ser Lys Val Asp Gly Ile Pro Met Glu Ile Tyr Val Val Ala Ile Met 275 280 285 Glu Asp Leu Asp Ile Val Ser Lys Gly Lys Ser Phe Lys Phe Gly Val 290 295 300 Ser Gln Asp Arg Ala Leu Ser Val Ala Gln Glu Leu Leu Asn Lys Ile 305 310 315 320 Leu Glu Ser Asp Lys Arg Lys Leu Arg Arg Val Gly Val Arg Leu Gly 325 330 335 Lys Ile Thr Lys Ser Ser Thr Leu Glu Asp Phe Leu His 340 345 <210> 14 <211> 376 <212> PRT <213> Artificial Sequence <220> <223> Nv Dpo4 <400> 14 Met Leu Ala Pro Pro His Arg Val Val Met His Ile Asp Phe Asp Tyr 1 5 10 15 Phe Phe Ala Gln Cys Glu Glu Val Arg Arg Pro Glu Ile Arg Gln Arg 20 25 30 Pro Val Leu Val Cys Val Phe Ser Gly Arg Thr Glu Asp Ser Gly Val 35 40 45 Val Ser Thr Ala Asn Tyr Val Ala Arg Lys Tyr Gly Val Lys Ser Gly 50 55 60 Ile Pro Ile Arg Val Ala Lys Ala Lys Leu Ala Gly Val Asp Asp Ala 65 70 75 80 Leu Phe Leu Pro Leu Asp Ala Ser Tyr Tyr Arg Gln Val Ser Glu Asn 85 90 95 Ala Met Ser Ala Ile Lys Glu His Ala Asp Val Phe Glu His Val Gly 100 105 110 Ile Asp Glu Cys Phe Ile Asp Val Ser Gly Arg Ala Asn Gly Arg Phe 115 120 125 Asp Ala Ala Glu Ala Leu Ala Arg Thr Val Lys Gln Arg Val Gln Glu 130 135 140 Arg Thr Lys Leu Thr Cys Ser Val Gly Val Ala Pro Asn Lys Met Leu 145 150 155 160 Ala Lys Ile Ala Ser Asp Tyr Asn Lys Pro Asp Gly Leu Thr Val Val 165 170 175 Arg Pro Glu Asn Ala Leu Gln Phe Val Ser Ala Leu Asp Val Asp Lys 180 185 190 Ile Pro Gly Ile Gly Pro Lys Thr Arg Asp Arg Leu Ala Glu Leu Gly 195 200 205 Val Lys Thr Ala Gly Asp Leu Ala Ser Phe Asp Leu Phe Arg Leu Ile 210 215 220 Lys Glu Phe Gly Lys Lys Thr Ala Thr Tyr Ile His Asn Ala Ala Lys 225 230 235 240 Gly Ile Asp Asp Glu Pro Val Met Glu Ser Ala Glu Gly Glu Arg Arg 245 250 255 Gln Gln Ile Met Arg Ile Val Thr Leu Lys Lys Asp Ala Gln Ser Ala 260 265 270 Glu Glu Met Tyr Ala Asp Leu Glu Glu Ile Cys Arg Asp Val Tyr Glu 275 280 285 Ser Ala Thr Glu Lys Lys Val Ala Phe Gly Ser Val Gly Ile Ile Leu 290 295 300 Val Leu Asp Asp Leu Glu Asn Val Thr Arg Ser Arg Gly Leu Lys Ala 305 310 315 320 His Ala Ser Ser Phe Glu Leu Leu His Ser Thr Ala Arg Ser Leu Leu 325 330 335 Asp Glu Ala Met Gly Glu Lys Lys Arg Ser Val Arg Arg Leu Gly Val 340 345 350 Arg Ile Ser Asp Phe Gln Asp Ser Ser Gly Gln Asn Thr Leu Phe Asp 355 360 365 Tyr Phe Thr Ala Arg Gln Gly Glu 370 375 <210> 15 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> Aa Dpo4 <400> 15 Met Pro Gln Phe Ile Ile Phe Ile Asp Phe Asp Tyr Phe Phe Ala Gln 1 5 10 15 Val Glu Glu Ile Leu Asn Pro Glu Ile Arg Glu Lys Pro Val Val Val 20 25 30 Cys Val Tyr Ser Gly Arg Thr Glu Thr Ser Gly Ala Val Ala Thr Ser 35 40 45 Asn Tyr Leu Ala Arg Lys Ile Gly Ile Lys Ser Gly Met Pro Leu Pro 50 55 60 Gln Ala Met Lys Ile Gly Lys Asp Arg Ala Val Phe Leu Pro Ile Arg 65 70 75 80 Lys Asp Ile Tyr Lys Glu Trp Ser Asp His Ile Met Asp Ile Ile Ser 85 90 95 Glu Tyr Ser Asp Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Ile 100 105 110 Asp Val Thr Asp Ser Cys Lys Asp Phe Asn Asp Ala Val Asn Thr Ala 115 120 125 Gln Lys Ile Lys Asp Glu Ile Tyr Asn Lys Thr Gly Val Arg Val Ser 130 135 140 Val Gly Val Ser Val Asn Lys Ala Ile Ala Lys Val Leu Gly Asp Met 145 150 155 160 Ala Lys Pro Asn Gly Ile Lys Ser Val Gly Pro Ser Glu Ile Gln Asp 165 170 175 Phe Leu Lys Gly Leu Glu Ile Ser Lys Ile Pro Gly Val Gly Lys Val 180 185 190 Ile Gly Gln Arg Leu Asn Asp Ala Gly Ile Lys Tyr Ile Thr Asp Val 195 200 205 Ile Asn Ala Asp Arg Asp Gln Leu Ile Asn Ile Ile Gly Thr Ala Lys 210 215 220 Tyr Asn Tyr Leu Tyr Asp Ile Ala Asn Asn Thr Tyr Asn Lys Pro Val 225 230 235 240 Thr Pro Arg Glu Lys Lys Asn Phe Gly Arg Tyr Met Thr Leu Pro Glu 245 250 255 Asn Thr Arg Asp Val Asn Ile Ile Ile Pro Tyr Val Lys Lys Ala Ile 260 265 270 Asp Ser Ala Tyr Glu Lys Ala Pro Gly Leu Pro Ser Glu Leu Ser Leu 275 280 285 Val Ala Ile Met Glu Asp Ile Ser Ile Leu Ser Arg Ser Tyr Thr Gly 290 295 300 Ala Arg Ile Asp Lys Asn Arg Ala Leu Glu Ile Ala Ile Gly Leu Leu 305 310 315 320 Asn Arg Ile Ile Asn Glu Asp Gln Arg Asn Ile Arg Arg Val Gly Val 325 330 335 Arg Leu Gly Lys Ile Thr Lys Asn Glu Thr Leu Asp Asp Phe Phe 340 345 350

Claims (14)

A protein domain having binding affinity to L-DNA.
The protein domain of claim 1, wherein the domain has at least 30% sequence homology with SEQ ID NO: 2.
The protein domain of claim 1, wherein the domain has at least 80% sequence homology with SEQ ID NO: 2.
An isolated L-DNA binding domain consisting of the amino acid sequence represented by SEQ ID NO: 1.
5. The isolated L-DNA binding domain of claim 4, wherein the isolated L-DNA binding domain is derived from a DNA polymerase.
The isolated L-DNA binding domain of claim 5 , wherein the DNA polymerase is DNA polymerase 4 (Dpo4).
5. The isolated L-DNA binding domain of claim 4, wherein the isolated L-DNA binding domain is a little finger domain of a DNA polymerase.
According to claim 4, wherein the isolated L-DNA binding domain is one selected from the group consisting of Arg (Arginine) at position 300, Lys at position 321 (Lysine), and Lys at position 339 (Lysine) of the amino acid sequence shown in SEQ ID NO: 2 An isolated L-DNA binding domain wherein at least one amino acid residue is not substituted or mutated.
A protein comprising the domain of any one of claims 1 to 8 .
The protein of claim 9, wherein the protein is applied to the preparation of antibody-drug conjugates.
The protein of claim 10, wherein the drug is selected from the group consisting of anticancer drugs, aptamers, antisense oligonucleotides, small interfering ribonucleic acids (siRNA, small interfering RNA) and micro RNA (miRNA).
A drug delivery system comprising the protein of claim 9 .
The drug carrier of claim 12; And comprising an anticancer agent, a pharmaceutical composition for the treatment of cancer.
The pharmaceutical composition of claim 13, wherein the anticancer agent is a DNA aptamer or an RNA aptamer.

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