WO2004069868A1 - Complex of telomere protein with guanine-quadruplex dna - Google Patents

Abstract

It is intended to provide a complex which is formed by a telomere protein TRF1 or TRF2 or a telomere protein fragment containing the DNA-binding domain thereof and a DNA having the guanine-quadruplex structure in the presence of a cation. Also, it is intended to provide a DNA capable of having the guanine-quadruplex structure. Use of the above-described DNA and complex makes it possible to analyze the mechanisms of aging and canceration and design drugs such as carcinostatic agents.

Description

Complex of telomere protein and guanine quadruplex helix DNA

 The present invention relates to a complex of a telomere protein and a DNA having a guanine quadruplex (hereinafter, referred to as “G-quadruplex”) structure. More specifically, the present invention relates to a telomere protein TRF1 or TRF2. G—Relates to a complex with DNA that has a quadruple helix structure.

 Light

Background art

 The telomere end of the chromosome consists of a telomere DNA sequence rich in guanine bases and is important to protect cells from external adverse effects and destruction. If this telomere cannot be maintained, it will eventually lead to cell death (apoptos is), so it is thought that this phenomenon can be applied to cancer treatment.

Recent studies suggest that telomere DNA can form a quadruple helix consisting of four strands [Figure 1], which may be involved in the structure of the telomere end. So far, G-quadruplex DNA structures have been created by in experiments with telomere sequences of several species, and X-ray structure analysis and NMR structure analysis have been performed. Many of the G-quadruplex DNAs whose structures have been analyzed are those whose structures themselves are energetically stable. In particular, G-quadruplex DNA formed by a telomere DNA sequence consisting of guanine (G) and thymine (T) Many helical DNA structures have been reported. These structures require monovalent metal cations to form the structure, and G-quadruplex DNA is formed by the cations of alkaline earth metals such as K + and Na ^+. [Fig. 1]. Since the human telomere sequence is a repeat sequence of TTAGGG and contains adenine (A), there have been few reports on the G-quadruplex DNA structure. Few structures Analyzed human G-quadruplex DNA structures are often based on NMR structure analysis, all of which are G-quadruplex DNAs coordinated with Na +, called anti-paral lel (antiparallel). is there. This is a group of G-rich chains antiparallel to each other.

Furthermore, last year, a paral lel (parallel) G-quadruplex DNA structure was reported that is fundamentally different from the G-quadruplex DNA structure already reported for the first time in human telomere sequences [Nature, Vol. 417, pp. 876-880]. This structure is a quadruplex crystal structure grown at a concentration close to the intracellular concentration using four consecutive human telomeric DNA repeat sequences, and K + is found in the crystal structure. Folding and appearance of DNA in the intramolecular quadruplex is fundamentally different from the G- quadruplex helical DNA structure containing a previous Na ^+. All four DNA strands are in a parallel relationship, and three trinucleotide loops bridging them are arranged like propellers outside the quadruplex core. An adenine residue in each TTA-bridged trinucleotide loop (T-TA 1 oop) is inserted back between two residues of thymine.

 However, it has not been known that DNA and a telomere protein having a G-quadruplex form a complex.

 The telomere DNA of mammalian, including human, vertebrates has a double-stranded TTAGGG repeat region ending in a 3 'single-stranded overhang (3'-overhang), which contains a telomeric repeat sequence binding factor ( The telomere protein hTRFl, which is a telomere repeat-binding agent (RFactor), specifically binds to RF2 as a homodimer [Figures 1 and 2]. RF1 binds to telomere binding proteins, Tankyrase and TIN2, and regulates telomere length. In addition, it has been suggested that hRapl localizes to telomeres via hTRF2 and regulates telomere length, but other functions have not yet been elucidated. In this way, the telomere end is regulated by various telomere-binding proteins as well as by tetramerase [Fig. 2].

Previous studies have shown that in cells with telomerase activity, telomere elongation occurs when hTF1 loses binding activity. When MRF2 loses its binding activity, the telomere becomes unstable and induces ATM (ataxia telangiectasia mutated) / p53 activity, leading to cell death (apoptos is). Thus, RF2 binds to the telomere terminus and protects telomere DNA from monitoring DNA repair mechanisms. As described above, the functions of these proteins in vivo are mainly that hTRF1 has a function to negatively regulate telomerase activity, and hTRF2 has a function related to telomere maintenance and protection. In addition, hTRFl has been observed to have the activity of folding double-stranded telomeric DNA in hTRF1, and hTRF2 is located between the double-stranded repeat sequence of the double-stranded throw-reference structure called the t-loop structure and the single-stranded protruding end. It has been observed that it is selectively located at the joint site of. At least six molecules of TTAGGG-3 'overhang are indispensable for maintaining at least the t_loop structure [Figure 3]. Telomere DNA is susceptible to external influences because it is located at the end of the chromosome. For this reason, it has long been predicted that telomeres may form a structure at the end in order to protect the telomere end. The t-loop structure formed by RF2 is a typical example [Fig. 3]. The formation of the t_loop is to protect the telomere end, and if it is not formed, it is thought that telomere end damage, telomere protruding end deletion, telomere fusion, etc. will occur, resulting in loss of telomere function. ing. Some papers have reported a model on the formation of this t_loop structure, but no actual proof has been found yet.

 hTRFl and MF2 both have a homodimer-forming dimerization domain (TRF Homology Domain: TRFH) in the center, and a DNA-binding domain (specifically recognizing double-stranded telomeric DNA at the C-terminus). PA-Ending Domain (DBD) exists [Figure 4]. The major difference is that they have an acidic domain and a basic domain at the N-terminus, respectively. Although the function of this region is not well understood, hTRF2 loses only its basic domain, reducing its ability to bind to telomere DNA and inactivating telomere function, leading to cell death. Comparing the amino acid sequences of the DNA-binding domains, the telomeric proteins RF1-DBD and RF2-MD have significant homology to the nuclear proto-oncogene product Myb proteins R1, R2, and R3. The three-dimensional structure of this protein consists of three helixes, where the hydrophobic amino acids present are almost completely conserved. These proteins have been shown to interact with DNA by the DNA binding domain alone, and the amino acid sequence homology between hTRF DBD and RF2-DBD is about 58%.

 An object of the present invention is to analyze the interaction between DNA having a G-quadruplex structure and the telomeric protein TRF1 or TRF2. Disclosure of the invention

 As a result of intensive efforts to solve the above problems, the present inventors have found that DNA having a G-quadruplex structure and a telomere binding protein TRF1 or TRF2 form a complex. It was completed.

 The gist of the present invention is as follows.

 (1) In the presence of cations,

Either TRF 1 or TRF 2 telomere protein or its DNA binding region A telomere protein fragment comprising:

A complex formed with guanine, a DNA having a quadruple helix structure.

 (2) The complex according to (1), wherein the cation is a sodium ion or a potassium ion.

 (3) The complex according to (1), wherein the telomere protein or a telomere protein fragment containing the DNA binding region thereof is selected from the group consisting of the following proteins (A) to (D) and protein fragments.

 (A) TRF 1 represented by the amino acid sequence of SEQ ID NO: 6

 (B) TRF 2 represented by the amino acid sequence of SEQ ID NO: 8.

 (C) a TRF1 fragment containing the DNA binding region of TRF1, represented by the amino acid sequence of SEQ ID NO: 5 (however, the first methionine residue may be deleted)

 (D) a TRF2 fragment containing the DNA binding region of TRF2 represented by the amino acid sequence of SEQ ID NO: 7 (the first methionine residue may be deleted)

 (4) The complex according to any one of (1) to (3), wherein the DNA having a guanine quadruplex structure has any one of the nucleotide sequences of SEQ ID NOs: 1 to 4.

 (5) The telomere protein or a telomere protein fragment containing the DNA binding region thereof is TRF1 or a TRF1 fragment containing the DNA binding region thereof, and the DNA having a guanine quadruplex structure is an antiparallel guanine fragment. The complex according to any one of (1) to (4), which has a quadruple helix structure.

 (6) The telomere protein or a telomere protein fragment containing the DNA binding region thereof is TRF2 or a TRF2 fragment containing the DNA binding region thereof, and the DNA having the guanine quadruple helix structure is composed of parallel guanine DNA. The complex according to any one of (1) to (4), which has a quadruple helix structure.

 (7) The complex according to (1), which is selected from the group consisting of the following complexes (i) to (iv).

(i) A complex of a TRF1 fragment containing the DNA binding region of TRF1 represented by the amino acid sequence of SEQ ID NO: 5 and a DNA having the nucleotide sequence of SEQ ID NO: 4 and having a guanine quadruplex structure

(ii) a TRF2 fragment containing a DNA binding region of TRF2 represented by the sequence consisting of the amino acid sequence of SEQ ID NO: 7, and a guanine quadruple helix having the nucleotide sequence of SEQ ID NO: 1 Complex with DNA

 (iii) A complex of a TRF2 fragment containing the DNA binding region of TRF2 represented by the amino acid sequence of SEQ ID NO: 7 and DNA having the nucleotide sequence of SEQ ID NO: 2 and having a guanine quadruplex structure

 (iv) A complex of a TRF2 fragment containing the DNA binding region of TRF2 represented by the amino acid sequence of SEQ ID NO: 7 and a DNA having the nucleotide sequence of SEQ ID NO: 3 and having a guanine quadruplex structure

 (iv) A complex of a TRF2 fragment containing the DNA binding region of TRF2 represented by the amino acid sequence of SEQ ID NO: 7 and a DNA having the nucleotide sequence of SEQ ID NO: 4 and having a guanine quadruplex structure

 (8) DNA having a base sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 13, 14, 15, and 16, and capable of forming a guanine quadruplex helix structure.

 (9) The DNA according to (8), which has a guanine quadruple helix structure.

 (10) The DNA according to (9), wherein the guanine quadruple helix is a parallel-chain guanine quadruple helix.

 (11) The DNA according to (9), wherein the guanine quadruple helix is an antiparallel guanine quadruple helix. As used herein, the term “guanine quadruplex” (G-quadruplex) refers to a structure in which four guanines (G) are stabilized by forming hydrogen bonds on a plane (see FIG. 1). (See reference).

 “A quaternary helix in a parallel chain” (G in a parallel chain—quadruplex helix) means that the chains of each of the four guanines (G) are all in the same direction (Dl, (See F2 and A4) This is because when the plane of four guanines (G) forming hydrogen bonds is viewed from above, the sugar monophosphate skeleton is located on this plane. Come or come down is the same. Here, the direction of the chain is the direction from the 5 'side to the 3' side of the sugar.

“An antiparallel guanine quadruplex” (antiparallel G—quadruplex) refers to a G—quadruplex other than the parallel strand G—quadruplex (Al in FIG. 5). , Bl, Cl, A2, B2, C2, D2, E2, B4, C4 and D4). The present invention relates to a DNA having a base sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 13, 14, 15, and 16 and capable of forming a guanine-quadruplex structure (hereinafter referred to as “the present invention”). DNA ”).

 The DNA of the present invention may be composed of any number of strands such as single strand, double strand, triple strand, quadruple strand and the like.

 Further, the DNA of the present invention may be a natural DNA or a modified nucleic acid. Modified nucleic acids include those having a triester bond (PS Miller, et al., Biochemistry, 16, 1988-1996 (1977)) and those having a methylphosphonate bond (PS Miller, et al., Biochemistry, 18, 5134). -5143 (1979)), those with phosphorothioate bonds (F. Eckstein, et al., Biochemistry, 22, 4546-4550 (1983)), those with phosphorodithioate bonds (J. Neielsen , et al., Tetrahedron Lett., 29, 2911-2914 (1988); WK-D. Brill, et al., J. Am. Chem. Soc., 111, 2321-2322 (1989)), phosphoramido Those with a date bond (B. Froehler, et al., Nucleic Acids Res., 16, 4831-4839 (1988)) and those with modified sugar moieties (F. Morvan, et al., Nucleic Acids Res., 16, 833-847 (1988)), polyamide nucleic acid (PE Nielsen, et al., Science, 254, 1497-1500 (1991)), complexed with Interichiri Yuichi (.L Letsinger, et. al., J. Am. Chem. Soc., 108, 7394-7396 (1981)) (M. Lemaitre, et al., Proc. Natl. Acad. Sci. USA, 84, 648-652 (1987); JP Leonetti, et al.) , Gene, 72, 323-332 (1988)) and the like, but are not limited thereto.

The DNA of the present invention can be prepared as follows. First, a single-stranded DNA (hereinafter referred to as “ssDNA”) having any one of the nucleotide sequences of SEQ ID NOs: 1, 2, 3, 4, 13, 14, 15 or 16 is synthesized by a known method. For example, synthesis is performed using a commercially available DNA synthesizer. Next, a buffer containing a cation (eg, NaCl, KCl, etc.) is added, and the ssDNA is heated at 80 to 95 ° C for 5 to 20 minutes and cooled to room temperature. After heating at 50 to 70 for 10 to 30 minutes and cooling to room temperature, G-quadruplex DNA is produced. Whether a DNA has a G-quadruplex structure can be confirmed by taking a circular dichroism (hereinafter sometimes referred to as “CD”) spectrum. Can be. In other words, when DNA has a parallel-stranded G-quadruplex structure, a positive maximum wavelength appears at 265 nm and a negative maximum wavelength at 245 nm, and DNA has an antiparallel G-quadruplex structure. , A positive maximum wavelength appears at 295 nm and a negative maximum wavelength appears at 265 nm.

 Further, the present invention provides a method for forming a telomere protein fragment containing either a TRF1 or TRF2 or a telomere protein fragment containing the DNA binding region thereof and a DNA having a guanine quadruplex structure in the presence of a cation. A complex is provided.

 The DNA forming the complex of the present invention may be composed of any number of chains such as a single strand, a double strand, a triple strand, and a quadruple strand.

 The DNA forming the complex of the present invention may be a natural DNA or a modified nucleic acid. Modified nucleic acids include those having a triester bond (PS Miller, et al., Biochemistry, 16, 1988-1996 (1977)) and those having a methylphosphonate bond (PS Miller, et al., Biochemistry, 18, 5134). -5143 (1979)), with phosphorothioate linkage (F. Eckstein, et al., Biochemistry, 22, 4546-4550 (1983)) with phosphorodithioate linkage (J. Neielsen, et al. , Tetrahedron Lett., 29, 2911-2914 (1988); W. Κ.-D. Brill, et al., J. Am. Chem. Soc., 111, 2321-2322 (1989)), Phosphoramidate binding (B. Froehler, et al., Nucleic Acids Res., 16, 4831-4839 (1988)) and modified sugar (F. Morvan, et al., Nucleic Acids Res., 16, 833) -847 (1988)), polyamide nucleic acid (PE Nielsen, et al., Science, 254, 1497-1500 (1991)), complexed with an intercalator (RL Letsinger, et al., J. Am. Chem. Soc., 108, 7394-7396 (1981)), high affinity for cell membranes Introduced a molecule (for example, poly-L-lysine, etc.) (M. Lemaitre, et al., Proc. Natl. Acad. Sci. USA, 84, 648-652 (1987); JP Leonetti, et al., Gene, 72, 323-332 (1988)) and the like, but are not limited thereto.

 TRF1 and TRF2 forming the complex of the present invention may be derived from any animal such as mammals (eg, human, mouse, monkey, etc.), birds, and the like.

Further, TRF1, TRF2 and their fragments that form the complex of the present invention may be wild-type or mutant. Examples of the wild-type TRF1 include those having the amino acid sequence of SEQ ID NO: 6. As a fragment of wild-type TRF 1, the sequence One having the amino acid sequence of No. 5 can be exemplified. Examples of the wild-type TRF2 include those having the amino acid sequence of SEQ ID NO: 8. Examples of fragments of wild-type TRF2 include those having the amino acid sequence of SEQ ID NO: 7. A mutant of TRF1, TRF2 or a fragment thereof comprises an amino acid sequence in which one or several amino acids have been deleted, substituted or added in any of the amino acid sequences of SEQ ID NOS: 5 to 8, In addition, peptides or proteins having the biological activity of TRF1, TRF2 or a fragment thereof can be exemplified.

 DNA having a guanine quadruplex structure can be prepared by the method described above.

The telomere protein of either TRF 1 or TRF 2 or a telomere protein fragment containing the DNA binding region thereof can be prepared by a known genetic engineering technique. For example, a DNA encoding a telomere protein of either TRF 1 or TRF 2 or a telomere protein fragment containing the DNA binding region thereof is prepared by a known method, or obtained from a preservation institution, and this DNA is obtained. After amplification with primers, incorporate into plasmid and express in host cells. The expressed telomere protein or a telomere protein fragment containing the DNA binding region thereof is purified by a known method. The guanine quadruplex structure of the DNA preserves the telomere protein fragment obtained in this manner or the telomere protein fragment containing the DNA binding region and the DNA having the guanine quadruplex structure. The complex of the present invention is formed by mixing under dripping conditions (for example, in a buffer used when preparing DNA having a guanine quadruplex structure). Complex formation can be confirmed using a Biacore (Biamolecular Interaction Analysis Core technology) device (Biacore). For example, when the DNA adopts a parallel-chain guanine-quadruplex structure, the telomere protein or the telomere protein fragment containing the DNA binding region is immobilized on the sensor chip, and then the telomere protein is ligated on the sensor chip. The above-mentioned protein or a fragment thereof may be contacted with DNA having a guanine-quadruplex structure. When the DNA has an antiparallel guanine-quadruplex structure, the DNA having the guanine quadruplex structure is immobilized on a sensor chip, and then the DNA is telomerized on the sensor chip. The protein or a telomere protein fragment containing the DNA binding region thereof may be contacted. A small amount of mass on the sensor-chip surface due to binding and dissociation between two molecules The change detected as SPR (Surface £ lasfflon £ esonaiice) signal, the time change of this signal is displayed as a graph called a Sensorgram, by analyzing this in analysis software, it is possible to determine the K _D values. K _D values are 1 0 ⁴ or less, preferably 1 0 ⁵ below, if more preferably 1 0 ⁶ or less, protein D NA is the specific binding, ie, the composite It can be said that it forms a body.

 Since telomeres are thought to be involved in the control of aging and canceration, use of the DNA and complex of the present invention to analyze the mechanism of aging and canceration and to design drugs such as anticancer drugs. It becomes possible. This description includes part or all of the contents as disclosed in the description and / or drawings of a Japanese patent application (ie, Japanese Patent Application No. 2003-32233), which is a priority document of the present application. BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 shows a G-quadruplex]) NA structure (G-Quadruplex, G-Quartet).

It is a G-quadruplex DNA structure formed by continuous Guanine-rich (G-rich) single-stranded DNA. M + represents a monovalent cation (eg, Li ⁺ , K +, Na +, N¾ +, etc.). Divalent cations such as Ca ²⁺ and Mg ²⁺ may be involved in the stabilization of the structure. The configuration of Guanine bases differs depending on the monovalent cation (M +) in the solution that coordinates to form the structure, and is roughly classified into a parallel (paral lel) structure and an antiparallel (anti-paral lel) structure. You. These structural differences can be distinguished by the absorption of the CD spectrum [Figure 10].

 Figure 2 shows a telomere control model in mammals.

 Tankyrase and TIN2 bind to hTRFl and regulate telomere length. Map 1 controls telomere length by localizing to telomere via image 2.

 Figure 3 shows the mechanism of mammalian telomere DNA.

 Mammalian telomeres are located at the ends of linear chromosomes, and at the 3 'end there are double-stranded TTAGGG repeats ending in single-stranded overhangs. It has been suggested that a t-loop structure is formed to maintain and protect telomere terminal function.

 FIG. 4 shows a functional map of the telomere protein and an amino acid sequence comparison. .

The telomeric proteins (liTRF1, hTRF2) are homozygous in the center as both functional domains. There is a dimer formation domain (TRF homology domain) that forms a mer, and a DNA-binding domain (PolyA-finding Domain: DBD) that specifically recognizes double-stranded DNA is present at the C-terminus. . The major difference between the two is that they have an acidic domain and a basic domain at the N-terminus, respectively. The function of this part is unknown, and loss of the basic domain of TRF2 inactivates telomere function, leading to cell death.

 Comparing the amino acid sequences of the DNA binding domains, the telomeric proteins MRF1 and RF2 have great homology to the nuclear proto-oncogene product Myb protein R1, R2, and R3. = The dimensional structure has three helices, and the hydrophobic amino acids are almost completely conserved. In addition, the homology between the humic acid sequences of hTRF1-DBD and hTRF2-DBD is about 58%. FIG. 5 shows various G-quadruplex structures.

 FIG. 6 shows a flow and an electrophoretic diagram of the purification method of the telomeric proteins hTRF1-DBD and MF2-DBD.

 hTRF l-DBD (amino acids 371-439 (SEQ ID NO: 5) having a methionine residue (Met) at the N-terminus; 70aa) in an E. coli BL2 UDE3) strain (Novagen) transformed with pET13a into which an expression portion was inserted It was expressed in large quantities. Moreover, hTRF2-DBD (amino acids 437-500aa (SEQ ID NO: 7) having a Met residue at the N-terminus; 64aa) is abundant in the zo / 'BL21 (DE3) strain [Novagen] into which pET23b containing the expressed portion has been introduced. Was expressed. In both cases, after recovering the E. coli, the protein was eluted with an ultrasonic crusher, and single-band telomere proteins could be isolated by cellulose phosphate column (P11) chromatography and gel filtration chromatography.

 FIG. 7 shows the N-terminal His-tag fusion + 10 aa extension.

The insert of RF2-DBD fused with N-terminal His tag with Nde I -BaniHI site was designed. _PET28a was used as the vector.

 FIG. 8 shows the amino acid sequence of the His-tag fused hTRF2-DBD.

 The sequence confirmed whether the His-tag fusion protein expression plasmid constructed by the ligation reaction was integrated as planned. As a result, expression systems for both the N-terminal His-tag fused hTRF2-DBD and the C-terminal His-tag fused hTRF2-DBD were created.

 FIG. 9 shows the final stage of His-tag fusion RF2-DBD.

Close comparison of hTRF2-DBD and His-tag fused RF2-DBD by Tricine SDS electrophoresis. Band detected by CBB staining. It is a concentrated sample after gel filtration. 6 Addition of xHis-tag Child volume increased. The molecular weight of each protein is as follows.

hT F2-DBD: 7539. 65 Da

 N-terminal His-tag fusion hTRF2-DBD: 9702. 97Da

 C-terminal His-tag fusion hTRFH) BD: 9259.49Da

 'Figure 10 shows parallel G-quadruplex DNA and anti-parallel G-quadruplex DNA by CD spectrum using trl2 +: 5'-TTAGGGTTAGGG-3' (SEQ ID NO: 1), which is the minimum recognition sequence for telomere protein. Shows discrimination from heavy helical DNA.

 (a) When K + is contained in the solution, a CD spectrum having a positive maximum at 265 nm and a negative maximum at 245 nm is observed.When Na + is contained, a positive maximum at 295 MI and a negative maximum at 265 nm are observed. A CD spectrum is observed. Similar spectra were observed with tr24 + antiparallel G-quadruplex DNA containing Na + in the solution. (B) Comparison of CD spectrum without valence cations (left) and comparison of CD spectrum of trl2—complementary to trl2 + in the presence of K + and Na + (right).

 Fig. 11 shows the 丽 R spectrum (one-dimensional spectrum) of trl2-, trl2 +, tr8 +, and trl2 +. Fig. 12 shows the NMR spectrum of the complex formed by trl2 + and TRF2-DBD. 1 shows an NMR spectrum (one-dimensional spectrum) of a complex formed by.

 FIG. 13 shows an NMR spectrum (two-dimensional spectrum) of a complex formed by trl2 + and TRF DBD.

 FIG. 14 shows an NMR spectrum (two-dimensional spectrum) of a complex formed by trl2 + and TRF2-DBD.

 FIG. 15 shows the prediction of the interaction site between trl2 + and TRF2-DBD. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described specifically with reference to Examples. These examples are for explaining the present invention, but do not limit the scope of the present invention.

[Example 1] Expression and purification of hTRFl-DBD and MRF2-DBD

Only the DNA-binding domains (iiTRF1-DBD and RF2-DBD) at the C-terminus of the telomere protein were respectively expressed from the Escherichia coli large expression system [Fig. 4]. hTRFl-DBD was obtained by extracting the pET13a plasmid from the RcoH \ (DE3) LysS strain for expression and transforming it into E. coli BL21 (DE3) strain to express it in large quantities.

 RF2-DBD was also expressed in large amounts in the ϋ / .BL2UDE3) strain into which the expression plasmid pET23b was introduced. In each case, after recovering the E. coli, the protein was eluted with an ultrasonic crusher, and the target protein was isolated by cellulose phosphate column chromatography and gel filtration chromatography.

 Next, we described each detailed method used in this study. experimental method

 1.1 Expression of hTRFl-DBD

 1) Change of expression system of hTRFl-DBD

Dr. Rhodes of MR C Laboratory at Gempridge University TRF DBD (Nucleic Acids Research, 1998, Vol. 26, No. 7, pp. 1731-1740, amino acid 371-439 with methionine residue (Met) at N-terminus) (SEQ ID NO: 5); pET13A (Gerchman, SE, Graz iano, V. and Ramakr ishnan, V. (1994) .Protein Express ion Purificat. 5, 242-251. This was introduced into the zo / BL2UDE3) strain to express hTRF2-DBD.

 ① Culture of E. coli BL21 (DE3) pLysS for hTRF DBD expression

First, LB medium (10 ml) was prepared in each of two clean test tubes, and sterilized by an autoclave (SANLS MLS-300, T0MY ES-315) [120 ° C, 15 to 20 minutes]. After cooling the medium to below 40-60 ° C in the Clean Bench, aseptically add the antibiotics 50 / ig / ml kana machine and 25 g / ml chloramphenicol, and add 1-8 (TC deep An R coli BL21 (DE3) pLysS strain (Novagen, Inc.) transformed with pET13a into which a portion coding for the sequence of the DNA binding domain of hTRFl stored in a DNA polymerase (Asahi Life Science ULT790-3J-A30) was inserted. Ltd.) were inoculated in an appropriate amount. the test tube was shaken 37 ° C 12 hours or more in the air bath bath. Thereafter, absorbance at a wavelength of 600nm with absorption spectrometer (Beckman Co. DU640) (0. D ₆₀₀₎ is At the point of 1.0 or more, the medium containing E. coli was collected and collected by centrifugation (5000 X g, 2 minutes) in a 1.5 ml eppen. Only the pellet (Escherichia coli) containing the hTRF1-DBD was recovered.

② Recovery of plasmid pET13a for expression of hTRFl-DBD For plasmid elution and recovery from E. coli, a Wizard Plus Minipreps DNA Purification system kit (Promega) was used. To the pellet from which the medium had been removed using sterile water in advance, the Cell Resuspention solution of the kit reagent was added, and the DNA / RNA degrading enzyme inhibition reaction was performed. Then, the cell membrane was dissolved with 200 / l of Cell lysis solution. The eluted nucleic acid, protein, impurities, etc. in E. coli were separated by adding 400 l of neutralization solution. This was centrifuged at IOOOOXg, and only the pET13a plasmid was eluted using a mini-column containing only the supernatant containing nucleic acids. At this time, since the E. coli strain was End A + strain, the enzyme was inactivated by adding 2 ml of a 40% isopropanol / 4.2 M guanidine hydrochloride solution.

③ For expression of hTRFl_DBD Ε · Transform into BL21 (DE3) and confirm expression

 Combine pET13a plasmids 1-21 with 60 l of the competent cell of the ZOZO'BL21 (DE3) strain, allow to stand still for 30 minutes while cooling accurately on ice, and give a heat shock at 42 ° C (about 45 seconds). E. coli was transformed by resting on ice for about 2 minutes. To this was added about 500 S0C medium, which had been pre-warmed and thawed at 37 ° C, and incubated for 1 hour or more in a thermostat at 37 ° C. The Escherichia coli grown in the S0C medium was then inoculated on an LB plate (kanamycin, chloramphenicol-resistant) previously warmed at 37 ° C., and kept at 37 for 24 hours until colony formation.

 Next, 100 ml of LB medium was prepared, and after autoclaving, 5 ml of LB medium was aseptically taken into two test tubes, and only one of the formed colonies was aseptically inoculated. An antibiotic [25 g / ml chloramphenicol, 50 g / ml kanamycin] was added thereto, and precultured at 37 ° C for 4 to 5 hours. Some glycerol stocks were made around 0, D 圆 = 0.5 — stored at 80 ° C. All of the remaining cultures were injected with IPTG at each step (0.4, 0.6, 0.8) of the 0.D fraction and used to confirm the expression of hTRFl-DBD.

2) Large scale cultivation of hTRF DBD expressing E. coli

 The plasmid pET13a expressing hTRFl-DBD was transformed by 1).

① Preculture

First, about 20-30 ml of LB medium was prepared in a clean 300 ml Erlenmeyer flask, and sterilized by an autoclave [120 ° C, 15-20 minutes]. The medium was cooled to 40-6 (TC or lower) in the Clean Bench, and then the antibiotics 50 g / ml Kanamashi: and 25 g / ml chloramphenicol were added aseptically. Created first from the display A colimi (DE3) strain (Novagen) transformed with hTRF DBD expression plasmid PET 13a was inoculated in an appropriate amount. The Erlenmeyer flask was shaken in an air bath at 37 ° C for 12 hours or more. After that, when the _{OD 6Q ()} at a wavelength of 600 nm reached 1.0 or more with an absorptiometer, all the medium containing E. coli was transferred to main culture.

 ② Main culture

A 3 L LB medium was prepared in a jar fermenter (AC-D-3, manufactured by Iwashiya ADM) for mass cultivation of Escherichia coli, and sterilized in an autoclave. After sterilization, the antibiotic 50 / g / ml kana machine and 25 g / ml chloramphenicol are added aseptically, and a few drops of decanol, a surfactant that serves as an antifoaming agent, are added at a temperature of 37 ° C. Incubated at 2 L / min air at 200 rpm. After inoculation of E. coli in the preculture, the cells were cultured at a temperature of 37 C, air at 5 L / min, and a rotation speed of 800 to 1000 rpm. 0. D ₆ wavelengths 600nm absorbance meter _{() ()} is from 0.6 to 0 7 i Sopropyl- Bok thio -. / 3 -D-garactopyranos ide hT down to a temperature of about 20 by the addition of (IPTG) Expression of Fl-DBD was induced. After that, when 0D reached the plateau, E. coli was recovered using an E. coli recovery buffer (50 potassium phosphate buffer [pH 8.0], 100 mM NaCl, ImM ethylenediaminetetraacetate (EDTA)). For recovery, use a centrifuge (Avanti JA25 manufactured by Beckman, himac CR 26H manufactured by HITACHI) at 4 ° C, 9000-12000 Xg for 20 minutes to collect Escherichia coli. The wet weight was measured and stored in a deep freezer at 180 ° C. TRF1-DBD expression was confirmed by Tricine SDS electrophoresis.

1. Purification of MRF1-DBD

 After recovering the E. coli, the hTRF DBD is eluted with an ultrasonic crusher, and gel filtration chromatography using cellulose phosphate column chromatography and HPLC (High performance liquid chromatography) [HiLoad ™ 26/60 Superdex ™ 30 prep. grade] and Phenyl Sepharose column [HiLoad ™ 26/10 Phenyl Sepharose High Performance] obtained by hydrophobic chromatography.

1) Ultrasonic crushing (Son i cat ion)

Gently thaw the lysate frozen and stored in a deep freezer at room temperature at room temperature, and add 1 protease inhibitor EDTA-iree and lOmM phenylmethylsul fonyl fluoride (PMSF) lml / metanol to the mixture. The cells were disrupted with a sonicator (S0NIFIER450 manufactured by BRANSON) to elute hTRFl-DBD. Crusher settings are: Time: Hold, Duty cycle (%): constant, Output control: The operation was performed at 20 to 30 so that the sample temperature did not exceed 4 ° C. I was frozen again in a deep freezer at 180 ° C for 3 minutes and 30 minutes to maintain the temperature limit and to disrupt the cells efficiently. The elution was confirmed by Tricine SDS electrophoresis.

 2) Centrifugal fractionation

 After crushing the cells, centrifugation was performed at 62,000 X g at 4 ° C for 90 minutes to remove the insoluble fraction in the E. coli sample. Thus, the supernatant containing hTRF1-DBD was recovered.

 3) Cation exchange chromatography

 [Principle] Ion-exchange chromatography is a method of separating molecules using the difference in electrostatic properties of molecules. Proteins have a pi (isoelectric point) where the sum of the charges is zero, and behaves as an anion at higher pH and as a cation at lower pH. Using this property, it is a method of purifying by binding or eluting to an ion exchange resin.

 [Operation] The supernatant collected in 2) was applied to a column filled with a cellulose phosphate resin [P11 manufactured by Whatman]. The column used about 20 g of P11 resin, washed with 0.5 M NaOH and 0.5 M HCl alternately added. First, add an appropriate amount of 0.5M NaOH to the P11 resin, stir for 2 minutes, allow the resin to settle by allowing it to stand for 3 minutes, decant only the supernatant, and reduce the ρΗ <11 by repeated washing with MilliQ. did. Next, 0.5M HC1 was added to cation-exchange all Na + to H +, which was also washed with MilliQ so that the pH finally reached> 3.

 After the cation-exchanged resin was packed in a dedicated column, it was replaced with the equilibration buffer overnight. Here, the sample supernatant was applied, and after all the samples were applied, the sample was washed once with an equilibration buffer. After the completion of the washing, the sample was eluted using a linear gradient method with a NaCl concentration gradient of 300 mM to 1100 ni. The eluate was collected in a fraction tube at a rate of 80 drops / tube (about 4 ml) using a fraction collector. At this time, the elution rate of the sample was set at about 50 ml / h. '

 A sample after application, a sample after washing (wash), and a fraction sample were flowed by Tricine SDS electrophoresis, and a sample showing hTRFl-DBD band was collected.

 4) Concentration

In order to improve the degradability by the following procedure, the sample collected in the fraction tube was centriprep [Amicon Centriprep3: 15 ml, molecular weight cut off 3000], Centricon [Amicon Centr icon3: volume 2 ml, molecular weight cut off 3000]. Glycerol was previously attached to the filter as a preservative, so it was washed with MilliQ before use. It was concentrated by centrifugation (4 ° C, 8000 X g).

 5) Gel filtration chromatography (Gel-fil trat ion Chromatography)

 [Principle] The column is packed with a gel with many pores, and when a protein is flowed through the column, a small protein passes through a larger amount of pores than a large protein, resulting in a long passage. Will pass through. Larger proteins do the opposite. In this way, separation can be performed using the “sieving phenomenon”, which can separate proteins according to their size and shape.

 [Operation] The concentrated sample was purified by gel filtration (HiLoadTM26 / 60 SuperdexTM30 prep grade) by HPLC (High performance liquid chromatography) (50 mM potassium phosphate buffer [ρΗ8.5], 1 M Ml). The hTRF l-DBD was eluted with Flow lml / min for 150 minutes. The target protein was recovered from the eluted sample by Tricine SDS electrophoresis.

 6) Hydrophobic Chromatography

 When contaminants cannot be removed in step 5), use a Phenyl Sepharose column.

(HiLoadTM26 / 10 Phenyl Sepharose High Performance ) was hydrophobic chromatographic I scratch using (Buf fer A; 50mM phosphate Kariumu buffer [. PH8 0], 1M noodles _{4) 2} S0 _4, 0. _05ηιΜ azide Sodium bromide (NaN ₃ ), Buf ferΒ; 50ι potassium phosphate buffer [pH 8.0], 0.05 mM NaN ₃ ).

 It flows through in about 25 minutes after addition to HPLC at a flow of 2-3 ml / min.

 7) Final sample adjustment

 HTRFl-DBD was confirmed to be isolated by Tricine SDS electrophoresis. The sample was concentrated with CentriBrep 'Centricon, lyophilized in a state where the sample was dissolved in a 50 mM potassium phosphate buffer [pH 7.0], 100 mM NaCl buffer, and stored at 120 ° C.

1.3 Mass cultivation of hTRF2-DBD expressing E. coli

A full-length clone of the TRF2 gene (Nature Genetics, Vol. 17, pp. 231-235.) Was donated by Dr. Lange at Rockefeller University, and the portion encoding the DNA binding domain region was cut out from this clone. This was incorporated into a pET23b plasmid (Novagen) to give a plasmid pET23b for expressing WRF2-DBD (amino acids 437-500aa (SEQ ID NO :) having a methionine residue (Met) at the N-terminus; 64aa). This was introduced to ^ ZO / BL21 (DE3) [Novagen] To express a large amount of hTRF2-DBD. The cells were cultured at 37 ° C. using 3 L of LB medium (0.6 g of the antibiotic ampicillin), and the expression was induced by adding isopropyl-ltio-j3-D-garactopyranoside (IPTG).

 1) Preculture

 First, about 20 to 30 ml of LB medium was prepared in a clean 300 ml Erlenmeyer flask, and sterilized by an autoclave [120 ° C, 15 to 20 minutes]. After the medium was cooled to 40 to 60 ° C or lower in the Clean Bench, 100 g / ml ampicillin, an antibiotic, was aseptically added thereto. Further, an ^ -coli BL21 (DE3) strain transformed with the RF2-DBD expression plasmid pET23b previously prepared from a deep freezer at 180 ° C was inoculated in an appropriate amount. The Erlenmeyer flask was shaken in an air bath at 37 "C for 12 hours or more. Then, when the OD at a wavelength of 600 nm reached 1.0 or more with an absorptiometer, the entire culture containing E. coli was fully cultured. Moved to

 2) Main culture

Three liters of LB medium was prepared in a jar ferment for large-scale cultivation of Escherichia coli, and sterilized in an autoclave. After sterilization, aseptically add 100 zg / ml ampicillin as an antibiotic, and add 2-3 drops of surfactant Adekinol, a defoaming agent, at a temperature of 37 ° C and air at 2 L / min. The plate was incubated at 200 rpm. After inoculation of pre-cultured E. coli, the cells were cultured at a temperature of 37 ° C, at an air rate of 5 L / min, and at a rotation speed of 800 to 1000 rpm. 0. D _MQ wavelength 600nm has IPTG was added to express induce WRF2-DBD down to a temperature 20 ° about C at 0.6 to 0.7 absorbance meter. Thereafter, when the _OD 6QD reached the plateau, E. coli was recovered using an E. coli recovery buffer (50 potassium phosphate buffer [pH 7.0], 100 mM NaCl, ImMEDTA). For recovery, E. coli was recovered by centrifugation at 9000-12000 X g for 20 minutes in a centrifuge at 4 ° C, and the wet weight was measured to estimate the expression level. saved.

Expression of TRF2-DBD was confirmed by Tricine SDS electrophoresis.

1.4 Purification of hT F2-DBD

 After recovering the Escherichia coli, MRF2-DBD was eluted with an ultrasonic crusher and obtained by cellulose phosphate column chromatography, gel filtration chromatography using HPLC, and hydrophobic chromatography on a Phenyl Sepharose column.

1) Ultrasonic crushing (Sonicat ion)

(1) Gently thaw lysate frozen in a deep freezer at 80 ° C at room temperature. To this was added 1 EDTA-free protease inhibitor and lOniPMSF lml / methanol, and the cells were disrupted with an ultrasonic disrupter to elute hTRF2-DBD. The setting of the crusher was as follows: Time: Hold, Duty cycle (%): constant, Output control: 20 to 30, and the operation was performed so that the sample temperature did not exceed 4 ° C. The temperature limit was maintained by freezing again in a deep freezer at _80 ° C for a few degrees for 30 minutes to efficiently disrupt the cells. The eluted RF2-DBD was confirmed by Tricine SDS electrophoresis.

 2) Centrifugal fractionation

 After crushing the cells, centrifugation was performed at 62,000 X g at 4 ° C for 90 minutes to remove the insoluble fraction in the E. coli sample. Thus, the supernatant containing hTRF2-DBD was recovered.

 3) Cation exchange chromatography

 The supernatant collected in 2) was applied to a column filled with a cellulose phosphate resin (P11). The resin subjected to the ion exchange was packed in a dedicated column, and was replaced with the equilibration buffer overnight. Here, the sample supernatant was applied, and after all the samples were applied, the sample was washed once with an equilibration buffer. After washing, the sample was eluted using a linear gradient method with a NaCl concentration gradient of 300 mM to 1100 mM. The eluate was collected in a fraction tube at a rate of 80 drops / tube (about 4 ml) using a fraction collector. At this time, the elution rate of the sample was set at about 50 ffll / h.

 A sample after application, a sample after washing (wash), and a fraction sample were flowed by Tricine SDS electrophoresis, and a sample in which an RF2-DBD band was observed was collected.

 4) Concentration

 In order to improve the decomposition performance by the following operation, the sample collected in the fraction tube was centriprep [Cenriprep3 by Amicon: capacity 15 ml, molecular weight cut-off 3000], Centricon [Centricon3 by Amicon: capacity 2 ml, molecular weight cut-off 3000] ]. Glycerol was pre-attached to the filter as a preservative, so it was washed with MilliQ before use. It was concentrated by centrifugation (4 ° C, 8000 X g).

 5) Gel filtration chromatography (Ge fi l trat ion Chrom tography)

The concentrated sample was purified by HPLC and gel filtration chromatography (50 phosphate buffer solution [pH 7.0], 1M NaCl). h-DBD was dissolved at a flow rate of 2 ml / min for 70 to 75 minutes. The elution was confirmed by Tricine SDS electrophoresis. 6) Final sample adjustment

 Tricine SDS electrophoresis confirmed that hTRF2-DBD was isolated. The sample was concentrated with CentriBrep and Centricon, lyophilized with the sample dissolved in a 50 mM potassium phosphate buffer [pH 7.0], 100 mM NaCl buffer, and stored at 120 ° C.

 * Concentration test

The concentration of telomere protein was determined using ultraviolet absorption. The molarity was determined according to Lambert-Beer's law. The protein detects the absorption by the side chains of Y (tyrosine) and W (tritophan) at 280 nm. This absorption depends on the content of Y and W. Determine the molar extinction coefficient £ ₂ .

Maximum absorbance wavelength of protein: 280 nm

£ ₂₈₀ = Trp number X £ T _ry + Tyr number X £ T _yr + Phe number X ερ ^

εχ _Γρ = 5500, ε _ΤγΓ = 1340, _{eph e} = 190

Lambert-Beer's law: Abs = ε ■ C · 1

 Absorbance (OD: Optical density), ε molar absorbance coefficient (1 / mol / cm), C: molar concentration (mol / 1), 1: cell width (cm)

Molar extinction coefficient of hTRFl-DBD, MRF2-DBD

ε ₂₈₀ = 4X 5500 + 2X 1340 + IX 190 = 24870 = 2.5Χ1θ4

Also, if εΤΓΡ: ⁵⁵⁰⁰ , £ Ty _r = 1340,

ε ₂₈₀ = 4 Χ 5500 + 2 Χ 1340 = 24680 = 2.5Χ1θ4

1.5 Experimental reagents

Table Η

 LB medium composition (3L)

 tryptone Peptone 30g, yeast extract 15g,

 NaCl 30g, IN NaOH 3ml, MilliQ 3L

 Autoclave sterilization

 M9 medium (3D

_{A: Na 2 HP0 4 38.2g,} KH 2 P04 9.0g, NaCl 1.5g,

4.5 g of ¹⁵ NH ₄ C1 (15N label), MilliQ 3L (autoclave sterilization);

B: 1M Mg0 ₄ 6.0ml (autoclave sterilization) C: 1M CaCl ₂ 300 1 (autoclave _{sterilization) D: C 6 H 12 0} 6 lOg ( filter sterilized) Table Bok 2

 3 Composition of X modifier

 6% SDS, 24% GlyseroK 12% 2-mercapto et anoL 0.3M Tris-HCl (pH6.8), 0.15% Bromophenol Blue Table 3

 Composition of Tricine SDS-PAGE

Separating gel

 46.5: 3.0 acrylamide solution 1.lml gel buffer 3.15ml

MilliQ 0.35ml

 Glycerol 1.7g

 10% APS 35 xl

 TEMED 3.75 l

 Stacking gel

 48: 1.5 acrylamide solution 0.225ml gel buffer 0.875ml

MilliQ 0.8ml

 10% APS 37.5 / il

TEMED 3.5xl

 Spacer gel

 48: 1.5 Acrylamide solution 0.45ml Gel buffer 1.125ml

MilliQ 0.675ml

10 PS 25 1

TEMED 2.5 zl 46.5: 3.0 acrylamide solution: acrylamide 6.5%, BIS 0.8% 48: 1.5 acrylamide solution: acrylamide 48, BIS 1.5% gel buffer · '· 2 · Tris-HCl (pH8.45), 0.3¾SDS Table H

 Tricine SDS electrophoresis buffer

Internal solution (Cathode): MilliQ 900ml, 1M Tricine (pH8.25) 99ml,

 Tris 11.97g, 10¾SDS 9.99ml

External solution (Anode): MilliQ 810ml, 2M Tris (pH8.9) 90ml Table 5

Composition of Kumasi-Brilliant-Bul-Stain (CBB stain) solution

2.5g / l Coomassie Brilliant Blue, 45% MethanoL 10% acetic acid

 Composition of decolorizing solution for CBB staining

5¾MethanoK 7.5% acetic acid Table 1-7

 Wizard Plus Minipreps DNA Purification system kit (Promega) DNA extraction kit using alkaline lysis method

Cell Resuspension solution

 50mM Tris-HCl [pH7.5]

 10ni EDTA

 100Ai / ml RNase A

Cell Lysis solution

 0. M NaOH

 1% SDS

Neutral izat ion 1.3 M potassium acetate [pH4.8]

40% isopropanol / 4.2M guanidine HC1

 Guanidine hydrochloride 66.9g I4.2 Dissolution with 2M HC1 Experimental results

 1.6 Expression of hTRFl-DBD

 1) Change of expression system of hTRF DBD

 ① Culture of E. coli BL21 (DE3) pLysS strain for expression of MRF-DBD

 Escherichia coli has a characteristic viscosity, which makes it difficult to handle and has a problem with the amount recovered. The expression level was low, probably because the cells themselves were old.

 ② Recovery of plasmid pET13a for expression of hTRFl-DBD

 It was found that the amount of plasmid that can be recovered is reduced unless the pellet is sufficiently dissolved in the cell lysis solution. The amount of plasmid recovered was increased by allowing time for the neutralization reaction with the neutralization solution. In order to express the endonuclease (EndA +) of the E. coli strain, the amount of recovered plasmid was reduced unless it was thoroughly inactivated with a 40% isopropanol / 4.2M guanidine hydrochloride solution.

 ③ Transform to E. coli BL21 (DE3) for hTRFl-DBD expression and confirm expression

 Transformation reactions were performed with accurate timing. Transformation was relatively smooth.

 2) Large scale cultivation of hTRF DBD expressing E. coli

All cultures were performed aseptically. The administration of IPTG at 0.6 D ₆₀₀ = 0.6 increased the expression of RFD DBD. At this time, the temperature was lowered from 37 ° C culture to around 20-25, and cultivated gently. In this way, hTRFl-DBD was transferred to the soluble fraction from the constant temperature culture at 37 ° C.

 The expression level of hTRFl-DBD in coli BL21 (DE3) was significantly higher than that in pLysS strain. In addition, E. coli has become easier to handle, so losses have been reduced.

 1.7 Purification of hTRFl-DBD

Purification was more complicated than hT F2-DBD. In particular, the presence of contaminants is a problem. When the pH is set to around neutral (pH 8.0 or lower), MRF DBD is co-precipitated, and all contaminants are removed by cation-exchange clotting matella and gel filtration chromatography. Some interaction It was difficult to remove. HTRFl-DBD could be isolated by increasing the number of gel filtration chromatography, decreasing the elution rate, or performing hydrophobic chromatography.

 E. coll BL21 (DE3) The amount of hTRFl-DBD expression in the coli BL21 (DE3) strain was significantly greater than in the pLysS strain, resulting in an increase in the final recovery.

 1.8 Mass culture of E. coli expressing hTRF2-DBD

All cultures were performed aseptically. Administration of IPTG at 0.6 at D ₆₀₀ = 0.6 promoted hTRF2-DBD expression smoothly. At this time, the temperature was lowered from 37 ° C culture to around 30 ° C and cultured. Almost all of the RF2-DBD was found in the soluble fraction. Despite the large amount of expression, the wet weight was about 12 g.

 1. 9 Purification of hTRF2-DBD

 The expression level of RF2-DBD was much higher than that of hTRF-DBD. Therefore, during electrophoresis, the concentration was so high that it was diluted several hundred times and applied. MRF2-DBD was completely isolated by cation exchange chromatography and gel filtration chromatography using a cellulose phosphate column.

 FIG. 6 shows the flow of the above experimental operation and the electropherograms of M1F1-DBD (8578.89 Da) and hTRF2-DBD (MW = 7539.65 Da) obtained.

[Example 2] Analysis of interaction between hTRFl-DBD, MRF2-DBD and G-quadruplex DNA (G-Quadrulex, G-Quartet)

experimental method

 2.1 Adjustment of His-tag fusion hTRF2-DBD

G - interaction with quadruple helix DNA and HTRF2- DBD, when by immobilized DNA side measured at Biacore without baseline stabilized, it was not possible to calculate the dissociation constant (K _D). It was also a question of whether the structure of the previously immobilized G-quadruplex DNA itself was always maintained. Therefore, considering the measurement of the interaction by immobilizing the protein side, a 6 X His-tag fusion protein with an extra 10 amino acid residues added to the N-terminus of hTRF2-DBD and the C-terminus opposite to it, respectively An expression system was first created.

 1) Creation of expression system for His-tag fusion tiTRF2-DBD.

① Culture of E. coli BL21 (DE3) for hT F2-DBD expression First, 10 ml of LB medium was prepared in a clean test tube and sterilized by autoclaving [120, 15 to 20 minutes]. After sterilization, cool the culture medium to 40 ~ 60 or less in (: ^ & 118611 (: 11), aseptically add 100 g / ml ampicillin as an antibiotic, and further deepen at 180 ° C. The W // BL21 (DE3) strain transformed with pET23b encoding the RF2-DBD amino acid sequence (SEQ ID NO: 5) stored in a freezer was inoculated in an appropriate amount. After that, the medium containing E. coli was collected when the absorbance _reached 0. D _6QQ of 1.0 or more, and the cells were collected by centrifugation (5000 xg, 2 minutes) in a 1.5 ml eppen. The supernatant was separated into a pellet and only the pellet (E. coli) containing the target hTRF2-DBD was recovered.

 ② Recovery of plasmid pET23b for expression of hTRF2-DBD

 For plasmid elution and recovery from E. coli, a Wizard Plus Minipreps DNA Purification system kit (Promega) was used. To the pellet from which the medium has been removed using sterile water in advance, add {1} 06111 ^ 8115 61 ^ 1011 solution 400 1 of the reagent, and react with the DNA / RNA degrading enzyme. To dissolve the cell membrane. The eluted nucleic acid, protein, impurities, etc. in E. coli were separated by adding 800 1 Neutralization solution. This was centrifuged at IOOOOXg, and only the pET13a plasmid was eluted using a mini-column containing only the supernatant containing nucleic acids. At this time, since the E. coli strain was End A + strain, the enzyme was inactivated by adding 2 ml of a 40% isopropanol / 42M guanidine hydrochloride solution.

 ③ Amplification of His-tag fusion hTRF2-DBD expression part (Insert)

 The ssDNA used as a primer for the target restriction enzyme site was purchased from Vex. Primers were designed to add an NdeI-BamHI site for the N-terminal His-tag. As a condition for primer design, the DNA encoding RF2-DBD should be placed 10 amino acids from the N-terminal His-tag, and the denaturation temperatures (Tm values) of 5'-primer and 3'-primer are almost the same. A 30 to 40 base sequence DNA was used.

5'-primer5 'primer for N-terminal His-tag

5, -CGCATATGGAAGACAGTACAACCAATATAACAAAAAAGCAG-3 '(SEQ ID NO: 9)

3'-primer: 3 'primer for N-terminal His-tag

5'-GATGGATCCTCAGTTCATGCCAAGTCTTTTCATG-3 '(SEQ ID NO: 10)

In addition, a primer for adding a Ndel-Sal I site for the C-terminal His-tag was designed. Step W

As a condition for the primer design, the denaturation temperature (Tm value) of 5'-primer and 3'-primer should be set so that the His-tag comes to the 9 amino acid extension from the DNA encoding hTRF2-DBD to the C-terminus. A 30 to 40 base sequence DNA was used to make the DNA identical.

5'-primer: For C-terminal His-tag 5, Primer

 5'-CGCATATGGMGACAGTACAACCAATATAACAMAAAGCAG-3, (SEQ ID NO: 11)

 3'-primer: 3 'primer for C-terminal His-tag

 5'-GATGTCGACGTTCATGCCAAGTCTTTTCATGGTC-3 '(SEQ ID NO: 12)

 These were used to amplify the target insert by PCR (GeneAmp PCR System2400, manufactured by PERKIN ELMER).

 処理 Treatment of hTRF2-DBD expression part (Insert) with restriction enzyme site

The restriction enzyme site amplified in (3) was treated with Nde I and BamHI. As the restriction enzyme activity buffer, H buffer (500 mM Tris-HCl, pH 7.5, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 1 M NaCl) was used. The reaction was allowed to occur overnight (over 10 hours) in a thermostat at 37 ° C.

 調整 Preparation of expression vector for His-tag fusion protein

PET28a [NdeI-BamHI] [Fig. 7] was treated with a restriction enzyme as a vector for expression of hTRF2-DBD for N-terminal His-tag. Also, for expression of hTRF2-DBD for C-terminal His-tag, pET21a [NdeI_BamHI] [FIG. 8] was used as a vector for restriction enzyme treatment. Both were purchased from Promega. Hbuffer (500 mM Tris_HCl, pH 7.5, 100 mM gCl ₂ , 10 mM Dithiothreitol, 1 M NaCl) was used as the restriction enzyme activity buffer. The reaction was allowed to occur overnight (over 10 hours) in a thermostat at 37 ° C.

 Pagarose electrophoresis and gel digestion

 After restriction enzyme treatment (④, ⑤), each insert and vector were subjected to 2% agarose electrophoresis to recover the target DNA (100 V, 。. Using ethidium bromide staining for staining with an ultraviolet illuminator. Detected.

Only the target band was separated from the agarose gel, and the gel digestion solution was added at only 11 per mg of agarose gel and dissolved well at 60-65. This was transferred to a mini column, and only the DNA was adsorbed on the filter overnight. Washing with ethanol precipitation was repeated at least three times and centrifuged, and the target DNA was eluted with distilled sterilized water. ⑦ Ligation reaction

 Increase the concentration of the recovered vector and insert on the insert side, insert: vector: mix at a ratio of 7 to 10: 1, add an equal volume of ligation reaction solution (manufactured by Toyobo Co., Ltd.), and heat at 16 ° C. Incubated for over 1 hour.

 R Transformation of R-coliPlus (DE3) -RIL strain for expression of His-tag fusion hTRF2-DBD for expression of RTR

 Mix pET28a plasmid 1-21 for N-terminal His-tag fusion hTRF2-DBD expression with R coli BL21- codonPlus (DE3)-co-immediate etent cel l 201 of RIL strain, and cool accurately on ice 30 After leaving still for a minute, a heat shock of about 42 ° C (approximately 45 seconds) was given, and the cells were again placed on ice for about 2 minutes to transform the target Escherichia coli. To this was added about 500 S0C medium, which had been pre-warmed and thawed at 37 ° C, and incubated in a thermostat at 37 ° C for 1 hour or more. The Escherichia coli grown in the S0C medium was then inoculated on an LB plate (containing 50 g / ml kanamycin) that had been previously warmed at 37 ° C and left overnight at 37 ° C until colonies formed. . Similarly, the C-terminal His-tag-fused hTRF2-DBD expression plasmid pET21a was also transformed into BL2 codonPlus (DE3) _RIL strain. However, the LB plate used was supplemented with 100 g / ml 7 ampicillin.

Next, 100 ml of LB medium was prepared and sterilized by autoclaving, and then 5 ml of LB medium was aseptically taken from each of the two test tubes, and only one of the formed colonies was aseptically inoculated. . An antibiotic 50 g / ml kanamidine for terminal His-tag was added at 100 xg / ml ampicillin), and precultured at 37 ° C for 4 to 5 hours. A part of the glycerol stock was prepared around 0.5 (D. _6m) = 0.5 and stored together. The remaining culture, each stage all _{0. D 6M (0. 6, 0.} 8) each Hi s by administering IPTG at - used for expression confirmation of tag fusion HTRF2_DBD. 2) Culture of N-terminal His-tag fusion hTRF2-DBD

 In order to use His-tag fusion MRF2-DBD as an immobilized ligand for the interaction measurement by Biacore, cultivation of about 400 ml of LB medium was performed. Culture and purification were carried out simultaneously on different LB media for N-terminal His-tag fused MRF2-DBD and C-terminal His-tag fused MRF2-DBD, respectively. ① Preculture

First, about 10 ml of LB medium was prepared in a clean test tube, and sterilized by autoclave [120 ° C, 15 to 20 minutes]. Cool the medium to below 40-60 ° C in the Clean Bench, To this, 50 g / ml kanamycin as an antibiotic was added aseptically. In addition, an appropriate amount of E. coli BL21 (DE3) strain _RIL (Novagen) transformed with the N-terminal His-tag fusion tiTRF2-DBD expression plasmid pET28a was prepared from a deep freezer at -80 ° C. . The test tube was shaken in an air bath at 37 ° C for 12 hours or more. After that, when the 0.D fraction at a wavelength of 600 nm reached L0 or more with an absorptiometer, all of the medium containing E. coli was transferred to main culture.

② Main culture

 400ml of LB medium was prepared in a clean 1L Erlenmeyer flask and sterilized in an autoclave [121 ° C, 15-20 minutes]. After sterilization, 50 g / ml kanamycin, an antibiotic, was aseptically added, and a drop of the surfactant Adekinol, a defoaming agent, was added to the plate and incubated at 37 ° C. After inoculation of pre-cultured E. coli, the cells were shake-cultured at a temperature of 37 ° C. 96 mg of IPTG was added at a wavelength of 0.6 to 0.7 using an absorbance meter at a wavelength of 0.6 to 0.7, and the temperature was lowered to about 25 ° C to induce the expression of N-terminal His-tag fusion RF2-DBD. . After that, when 0D 删 reached a plateau, E. coli was recovered using E. coli recovery buffer (50 mM potassium phosphate buffer [pH 7.4], 150 mM NaCl, ImM EDTA). Use a centrifuge for recovery. C, and centrifuged at 9000-12000 X g for 20 minutes to collect E. coli. After measuring the wet weight for estimation of the expression level, the cells were stored in a deep freezer at 180 ° C. Finally, the expression of N-terminal His-tag fused hTRF2-DBD was confirmed by Tricine SDS electrophoresis.

3) N-terminal His-tag fusion RF2-DBD purification

 After recovering Escherichia coli, His-tag fusion RF2-DBD is eluted with an ultrasonic crusher, and gel filtration chromatography using cellulose phosphate column chromatography and HPLC [HiLoad ™ 26/60 Superdex ™ 30 prep grade] Was obtained. '

 ① Ultrasonic crushing (Sonicat ion)

 (1) Gently thaw the lysate frozen in a deep freezer at 80 ° C at room temperature, add 1 protease inhibitor EDTA-free and 1 ml of lOmM PMSF / methanol, and disrupt the cells with an ultrasonic disrupter. Then, N-terminal His-tag fusion 1F2-DBD was eluted. Crusher settings are: Time: Hold, Duty cycle (): constant, Output control: 20-30, sample temperature 4 The operation was performed so that it did not exceed C or higher. The temperature was maintained by freezing again in a deep freezer at 180 degrees for two or three degrees for 30 minutes to efficiently disrupt the cells.

② Centrifugal fractionation After disruption of the cells, centrifugation was performed at 62000 × g, 4 ° C. for 90 minutes to remove the insoluble fraction in the E. coli sample. Thereby, the supernatant containing the N-terminal His-tag-fused hTRF2-DBD was recovered.

③ Cation exchange chromatography

 The resin stored at 4 ° C as stock was packed in a special column, and was replaced overnight with an equilibration buffer (50 mM potassium phosphate buffer [pH 7.4], ΙΟΟπιΜ NaCl, ImM EDTA). . Here, the supernatant collected in (2) was applied to a column filled with cellulose phosphate resin (P11), and all samples were applied and washed once with an equilibration buffer. After washing, the sample was eluted by the step-by-step method using NaCl concentrations of 200, 300, 400, 500, and 600. Each sample was collected in a 50 ml conical tube.

 After Tricine SDS electrophoresis, a sample after application, a sample after washing (wash), and a fraction sample were flowed, and a sample in which a band of NTR-His-1ag-fused hTRF2-DBD was observed was collected.

 ④ Concentration

 In order to increase the resolution by the operation of ⑤, the sample collected in the fraction tube was centriprep [Amkon Centr iprep3: volume 15 ml, molecular weight cut off 3000], Centricon [Amicon Centricon3: volume 2 ml, molecular weight cut off 3000] It was carried out using. Glycerol was pre-attached to the filter as a preservative, so it was washed with MilliQ before use. It was concentrated by centrifugation (4, 8000 X g).

 ⑤Gel filtration chromatography (Ge filtration Chromatography)

 The concentrated sample was purified by gel filtration (HiLoadTM26 / 60 SuperdexTM30 prep grade) chromatography (10 mM HOPES buffer [pH 7.4], 100 mM NaCl, lmM EDTA). The N-terminal His-tag fusion RF2-DBD was eluted at a flow rate of 2 ml / min for 100 minutes [Fig. 9].

4) Cultivation of C-terminal His-tag fusion hTRF2-DBD

① Preculture

First, about 10 ml of LB medium was prepared in a clean test tube, and sterilized by autoclave [120Τ, 15 to 20 minutes]. After the medium was cooled to 40-60 t: or less in Clean Benc, the antibiotic 100 / ig / ml ampicillin was aseptically added thereto. Further, the plasmid pET21a, which generated the C-terminal His-tag fusion hTRF2-DBD previously prepared from a deep freezer at 180 ° C., was transformed: 'BL21 (DE3) strain-RIL was inoculated in an appropriate amount. Air this test tube Shake in a bath at 37 ° C for 12 hours or more. After that, when the _{0.6D of 0.6D at} 600 nm _reached 1.0 or more with an absorptiometer, all of the medium containing E. coli was transferred to main culture.

② Main culture

400ml of LB medium was prepared in a clean 1L Erlenmeyer flask and sterilized by autoclaving [12TC, 15-20 minutes]. After sterilization, 100 / _g / ml ampicillin, an antibiotic, was added aseptically beforehand, and a drop of the surfactant Adekinol, a defoaming agent, was added, followed by incubation at 37 ° C. After inoculation of pre-cultured E. coli, the cells were shake-cultured at a temperature of 37 ° C. Photometer 0. D _6M wavelength 600nm is 0.5 at 6-0. Seven by the addition of IPTG 96mg to express induce C-terminal Hi s-tag fusion WRF2- DBD down to a temperature 25 ° C extent in . After that, when 0D 咖 reached the plate, E. coli was recovered using E. coli recovery buffer (50 mM potassium phosphate buffer [pH 7.4], 150 mM NaCl, ImM EDTA). For recovery, use a centrifuge to centrifuge at 9000-12000 X g for 20 minutes at 4 ° C to collect the E. coli, measure the wet weight to estimate the expression level, and place in a deep freezer at 180 ° C. saved. Finally, expression of hTRF2-DBD fused with C-terminal His-tag was confirmed by Tricine SDS electrophoresis.

5) Purification of C-terminal His-tag fusion RF2-DBD

 As in the purification of the N-terminal His-tag fusion RF2-DBD, after recovering the E. coli, the C-terminal His-tag fusion RF2-DBD was eluted with an ultrasonic crusher, and the cellulose phosphate column chromatography and HPLC were used. Obtained by gel filtration chromatography.

 ① Ultrasonic crushing (Son i cat ion)

 — Gently thaw the lysate frozen in a deep freezer at 80 ° C at room temperature, add 1 protease inhibitor EDTA-free and 1 ml of lOmM PMSF / methanol and disrupt the cells with an ultrasonic disrupter. The C-terminal His-tag fusion hTRF2-DBD was eluted. The setting of the crusher was as follows: Time: Hold, Duty cycle (%): constant, Output control: 20 to 30, and operation was performed so that the sample temperature did not exceed 4 ° C. The temperature was maintained by freezing again in a deep freezer at 180 ° C for a few degrees for 30 minutes to efficiently disrupt the cells.

 ② Centrifugal fractionation

After crushing the cells, centrifugation was performed at 62,000 X g at 4 ° C for 90 minutes to remove the insoluble fraction in the E. coli sample. As a result, the supernatant containing the C-terminal His-tag fusion hTRF2-DBD was recovered. ③ Cation exchange chromatography

 The resin stored at 4 ° C as stock was packed in a special column, and was replaced overnight with an equilibration buffer (50 potassium phosphate buffer [PH7.0], lOOmM NaCl, ImM EDTA). . Here, the supernatant collected in (2) was applied to a column filled with cellulose phosphate resin (P11), and all samples were applied and washed once with an equilibration buffer. After the washing was completed, the sample was eluted by a step-by-step method using a buffer solution having a NaCl concentration of 200, 300, 400, 500, or 600 mM. Each sample was collected in a 50 ml conical tube.

 A sample after application, a sample after washing (wash), and a fraction sample were flowed by Tricine SDS electrophoresis, and a sample in which a C-terminal His-1ag-fused hTRF2-DBD band was observed was collected.

 ④Concentration,

 In order to increase the resolution by the operation of ⑤, use the sample collected in the fraction tube with Centriprep [Amicon Centriprep3: 15 ml, molecular weight cut off 3000], Centricon [Amicon Centricon3: 2 ml, molecular weight cut off 3000] I went. Glycerol was used as a preservative beforehand, so it was washed with MilliQ before use. The mixture was concentrated by centrifugation (4, 8000 × g).

 ⑤Gel filtration chromatography (Ge filtration Chromatography)

The concentrated sample was purified by HPLC and gel filtration chromatography (1 OmMHEPES buffer [PH7.4], lOOinM NaCl, ImM EDTA) ₀ C-terminal His-tag fusion hTRF2-DBD was eluted with Flow 2 ml / min for 100 min. [Figure 9].

 2. Confirmation of G-quadruplex DNA structure by circular dichroism (CD) spectrum measurement

 It is important to know the state of the sample when applying to Biacore. In particular

G-quadruplex DNA has a different structure depending on the buffer in which DM is dissolved, and therefore, there has been a problem that it has to be unified to measure the interaction. A CD measurement was made to confirm this easily.

 1) Preparation of G-quadruplex DNA

 The purchased ssDNA (Vex) was gently cooled to room temperature at 94 ° C for 10 minutes, and then

G-quadruplex DNA was prepared by annealing again for 15 minutes. Adjustment of K + buffer (10 HEPES [pH 7.4], 100 mM KC1) to create G-quadruplex DNA with parallel structure, annealing did. In addition, in order to prepare antiparallel G-quadruplex DNA, Na ⁺ buffer solution (lOmMHEPES [pH7.4], lOOmM NaCl) was prepared and annealed. The pH was adjusted using a base (KOH, NaOH) adjusted to the cation of each buffer solution.

The formed G-quadruplex DNA forms tr12 +, which has the minimum recognition base sequence of double-stranded telomere DNA, and tr24 +, TT-Aloop, which is twice the sequence, to measure the interaction with Biacore. Tr8 +, which cannot be used, and tr22 +, whose structure is already known, were used. The sequence, tr8 +: 5 '-TAGGGTTA- 3' ( SEQ ID NO: 13), trl2 +: 5 '-TTAGGGTTAGGG-3' ( SEQ ID NO: 1), tr22 +: 5 ' -AGGG (TTAGGG) 3 -3' ( SEQ ID NO: 3), tr24 +: 5,-(TTAGGG) ₄ -3 '(sequence number 4) We tried to make both parallel and antiparallel structures. In addition, in order to examine whether there is a sequence that easily forms a stable G-quadruplex DNA structure from the CD spectrum, 5 'G-trl2 +: 5'-GTTAGGGTTAGG-3' (SEQ ID NO: 14), trl3 used in dsDNA + And 5 'C-trl3 +: 5'-GTTAGGGTTAGGG-3' (SEQ ID NO: 2) and 5'-CTTAGGGTTAGGG-3 '(SEQ ID NO: 15), and double its sequence tr26 +: 5'- ( TTAGGG) JT-3 '(SEQ ID NO: 16) and 5' C-trl7 + with a nucleotide unrelated to the telomeric sequence added to the 5 'side of trl3 +: 5'-CATCATTAGGGTTAGGG-3' (SEQ ID NO: 17) and G -CD measurement was performed by forming quadruplex DNA.

 2) Structural confirmation by CD spectrum measurement

 The measurement was performed using a measuring instrument J-720 manufactured by JASCO Corporation, and the CD spectrum was measured using a program for baseline measurement and standard measurement. The measurement conditions are: set temperature 25 ° C (298K), measurement wavelength 190 to 320 (displayed as 220 to 32011111), resolution 0. lnm, wavelength sweep speed 20nm / min, integration frequency 4 times, response lsec, bandwidth L0nm Sensitivity was set to 50mdeg.

2.3 Measurement of interaction between hTRFl-DBD and hTF2-DBD and G-quadruplex DNA by BiacoreCarboxymethyldextran is coated in advance on the Au surface on the sensor chip, and NTA [N- (5- amino-l-carboxypentyl) iminodiacetic acid)] is covalently immobilized. Here, the buffer was chelated, and the His-tag fusion protein was immobilized specifically through the buffer. .

1) Adjusting the running buffer

As the Runninig buffer, an eluent buffer prepared by diluting the concentration of 3 EDTA to about 50 M from HBS-EP, which is a standard buffer of Biacore, was used after filtration and deaeration. Measuring When the buffer was replaced, the dissociation constant K _D under that condition was derived.

 2) Adjustment of ligand

 In this experiment, the protein side of the 6XHis-tag fused hTRF2-DBD was immobilized. This principle, like the principle of Ni column chromatography purification, utilizes the principle that the NTA on the chip acts as a chelating agent and the His-tag fusion protein specifically binds via Ni. The His-tag fusion protein was replaced with running buf fer from the purification buffer and used for the immobilization procedure. At the time of application, manual injection was used.

 3) Analytical adjustment

 The analyte concentration depends on the molecular weight of the analyte and the strength of the affinity, so it is better to adjust it appropriately.However, since the interaction between protein and G-quadruplex DNA is weak, the lOOuM Was adjusted with Running buffer.

 4) Sensor one chip selection

Interaction between RF2- DBD and G- quadruplex helical DNA, compared to the case of dsDNA, fried interaction weak, Sensorgram that Michibikeru the dissociation constant K _D values in the interaction experiments using SA chi is not obtained, et al. Was. Therefore, NTA chip was selected by immobilizing the protein side. The chips were stored at 4 ° C using the infiltration method.

 5) Fixing NTA chip

 First, the operation Prime was performed three times. After replacement with HBS-EP buffer and stabilization, substitution with Eluent buf fer was performed and stabilized. Then, Ni buf fer of about 500 M was applied at a flow rate of 20 1 / min to chelate the Ni on the NAT on the sensor chip and allowed to stand until it stabilized. After stabilization, a His-tag fusion protein RF2-DBD having 6 bases Histine was applied and immobilized on the sensor chip. At this time, an appropriate amount was immobilized using a manual injection. The protein was allowed to stabilize as an interaction measurement baseline, and protein-DNA interaction was measured. G-quadruplex DNA whose structure was previously determined by CD measurement was applied as an analyte.

6) Sensorgram analysis (K _D value determination)

From the resulting Sensorgram, performs fitting with analysis software B Iacore accessory using nonlinear least-squares method (BIAevalut ion vers ion 3), was determined K _D values in the condition of each buffer. 2.4 Experimental reagents

Table 2-1

Restriction enzyme treatment

Restriction enzyme reaction conditions

10 X activity buffer

 Restriction enzyme treated DNA

 Sterile distilled water * After increasing the volume to 0 ^ 1, incubate at 37 ° C for 8-10 hours

* Sterile distilled water was used by sterilizing Mil 1 iQ with an autoclave. Table 2-2

 DNA amplification with KOD-Plus-DNA Polymerase

Table 2-3

Adjusting sequence samples

Table 2-4

 Eluent Buf fer: Standard buffer for immobilizing NTA chip

Anti-parallel G-quadruplex DNA

 lOmM HEPES [ρΗ7.4], 150ni NaCK 50 M EDTA, 0.005% Tween20

For parallel G-quadruplex DNA

 lOmM HEPES [pH 7.4], 150raM KC1, 50 EDTA, 0.005% Tween20 Table 2-5

 Ni solution [dissolved in Eluent Buffer]: Buffer for binding His-tag fusion protein

For antiparallel G-quadruplex DNA

Eluent buf fer, 500 M NiCl ₂

For parallel G-quadruplex DNA

Eluent buf fer, 500 M NiCl ₂ Table 2-6

 Regeneration solution: His-tag fusion protein regeneration buffer

For antiparallel G-quadruplex DNA ※ !! Adjustment → 1M NaOH

 lOmM HEPES [pH 7.4], 150 mM NaCK 350 mM EDTA, 0.005% Tween20

For parallel G-quadruplex DNA ※! ) !! Adjustment → 1M 0H Table 2-7

 Dispensor Buf fer: Buffer for removing unwanted metal ions and stabilizing the sensor chip For antiparallel G-quadruplex DNA

 HBS-EP

For parallel G-quadruplex DNA

 HBP-EP Table 2-8.

Interaction ・ Dissociation constant measurement buffer For antiparallel G-quadruplex DNA

 lOmM HEPES [pH 7.4], 150 mM NaCL 0.005% Tween20

For parallel G-quadruplex DNA

 lOni HEPES [pH 7.4], 150mM KCK 0.005% Tween20 Experimental result

 The interaction could be measured by Biacore with the His-tag fusion protein immobilized. The immobilized RF2-DBD was a 6 x His-tag fusion protein with an extra 10 amino acid residues added to the N-terminus and the opposite C-terminus, and there was no difference in the interaction using either. .

 2.5 Adjustment of His-tag fusion RF2-DBD

 The plasmid was recovered from Escherichia coli expressing MRF2-DBD, but the recovered amount was less than expected. For this reason, primers to which the insert restriction enzyme site of MRF2-DBD was added were purchased from Vex and amplified by the PCR method. The temperature conditions at that time were adjusted according to the denaturation temperature of the used primer. The amplified restriction enzyme sites could be treated with Nde I and BamHI. Similarly, pET28a [Nde I -BamH I] for expression of hTRF2-DBD for N-terminal His-tag, and pET21a [Nde I -BamH I for expression of 丽 2-fraction for C-terminal His-tag I] was used as a vector for restriction enzyme treatment. The target band was recovered by 2% agarose electrophoresis and subjected to gel digestion. Staining was carried out with ethidium mouth-mide staining using an ultraviolet illuminator. These were subjected to a ligation reaction, and both were able to be transformed into R coli BL21-codonPlus (DE3) -RIL strain.

For culture and purification, N-terminal His-tag-fused hTRF2-DBD and C-terminal His-tag-fused hTF2-DBD were simultaneously used in separate LB media. Regarding protein expression, it was most appropriate to add IPTG at O.D _{6D ()} = 0.6 to 0.7. The addition of IPTG and the culture at a reduced temperature of 25 promoted the solubilization of the fusion protein. His-tag fused RF2-DBD was eluted by sonication and purified using cellulose phosphate column chromatography and gel filtration chromatography. At this time, the eluted salt concentration in the cellulose phosphate columnography was increased to about 400 ffiM by fusing His-tag to the protein. The recovered protein has no problem in solubility even if it is handled at room temperature. N-terminal His-tag fusion in 400 ml culture The MRF2-DBD was about 450 / iM / lml, and the C-terminal His-tag fused hTRF2-DBD was about 200 200 / 1Κ1. As a result, the Ν-terminal His-tag fusion protein expressed more than the C-terminal His-tag fusion protein and could be recovered.

 2.6 Confirmation of G-quadruplex DNA structure by circular dichroism (CD) spectrum measurement

 As for the G-quadruplex DNA, the structure formed differs depending on the buffer in which the DNA is dissolved, so the structure was confirmed by CD measurement. The purchased ssDNA was gently cooled to room temperature at 94 ° C for 10 minutes, and then reannealed at 60 ° C for 15 minutes to produce G-quadruplex DNA. The annealing of the G-quadruplex DNA structure in this experiment was uniformly performed at this temperature. The formed G-quadruplex DNA was measured using a measuring instrument J-720 manufactured by JASCO Corporation.

As a result, G-quadruplex DNA from human telomere sequence: ① formed parallel G-quadruplex DNA in the presence of K +, while formed antiparallel G-quadruplex DNA in the presence of Na +. [Fig. 10], ② Stabilization of the formed structure depending on the concentration of the monovalent cation-containing salt used in the ring, and ③ Low temperature after the ring The structure formed by aging under a certain time is stabilized, and the concentration of ssDNA at the time of cycling determines the structure formed, which is particularly the case with long telomere DNA sequences. -What was often observed after ringing, and finally, the antiparallel structure was destabilized by the coexistence of the divalent cations Ca ²⁺ and Mg ^2+. Is there any property such as no change or other phenomenon seen as a result of the CD spectrum? Found was.

2.7 Interaction measurement of hTRFl-DBD and TRF2-DBD with G-quadruplex DNA using Biacore Ni-to-NTA covalently bonded to carboxymethyldextran previously coated on Au surface on sensor chip The buffer was chelated, via which the His-tag fusion protein was specifically immobilized.

 A CD spectrum was taken in advance to confirm that the G-quadruplex DNA structure was not disrupted by running buf fer, and used as an applied sample after confirmation.

In this experiment, the protein side of the 6XHis-tag fusion RF2-DBD was immobilized by manual injection. The baseline was easily stabilized by the time of analysis application. However, the baseline after addition of the analyte became unstable with each measurement, and this problem was solved by lowering the flow rate of Biacore. The concentration of the added analyte is predicted from the molecular weight of the analyte and the strength of the analyte, and the interaction of protein-G-quadruplex DNA is experimental. Since it was found that the measurement was weak, the measurement was performed in units of / M. As a result, we were able to measure the interaction that led to the value.

From the obtained Sensorgram, fitting was performed with the analysis software (BIAevalution version 3) attached to Biacore using the nonlinear least squares method, and the I ( _D value under each buffer condition was determined.

HTRF2-DBD from K _D values obtained are parallel G than antiparallel G- quadruplex helix DNA of sstrl2 + - it was found to strongly interact with quadruple helix DNA. In addition, it was found that sstr24 + interacts more strongly with antiparallel G-quadruplex DNA than sstr22 + antiparallel G-quadruplex DNA. This sstr24 + antiparallel G-quadruplex DNA structure was observed to interact strongly with hTRF DBD. Therefore, for hTRFHDBD, the dissociation constant (K _D ) could be derived using the SA chip [Table 3]. However, as for MRF2-DBD, the binding was unstable, and the baseline was not stabilized. Table 4 shows the results.

Table 3

Results of measurement of interaction between telomeric protein TRF2-DBD and G-quadruplex DNA and dissociation constant K _D

ssDNA G-quadmplex structure¾αα dragon J¾t K _D (M) ka (1 / Ms) kd (1 / s) K _D (M) ka (1 / Ms) kd (1 / s) tr8 + parallel 150mMKCI no data no data ^{no data 8.19X10 "4 4.67 x10 2} 5.32 X 10" 2 tr12 + parallel 150mMKCI no data no data no data 2.45X 10- 5 30.6 X10 4 7.49X10 one ¹

75m KCI no data no data no data 7.53x10 one ^{6 3.79 X 10 3 2.85X 10- 2} anti-parallel 150mMNaCI no data no data no data 2.23X10 one ³ 3.97 X 10 ^{2 8.88x10} one ¹

75mMNaCI no data no data no data 4.72 X 10 "4 1.00 X 10 3 4.72X 10- 1 tr22 + ant door parallel 150m NaCI no data no data no data 2.35x10 one ^{4 2.10 X10 3 4.93X 10- 1 tr24} + anti-parallel 150mMNaCI 4.27 X 10 " ⁸ 2.62 X 10 ⁵ 1.12X 10 7.56X10 1 ⁵ 9.90 x 10 ³ 7.48X10 ¹ lOOmMNaCI 5.03 X 10" ⁸ 6.52 X10 ⁴ 3.28X10 1 ³ 4.06 X 10 " ⁶ 3.08 X 10 ³ 1.25 X 10" ^Two

Table 4

Dissociation constant (K _D ) between telomere protein and G-quadruplex DNA by Biacore

Interaction between TRF2-DBD and G-quadruplex DMA

 * KCI was used for the parallel structure.

 * * 75 mM NaCI (KCI for parallel structure) was used in the running buffer.

 *** The SA chip was used for Biacore measurement.

Consideration

 Telomere DNA at the telomere end of eukaryotes can form a quadruplex consisting of four strands in /, [Figure 1], which is thought to be involved in the structure of the telomere end. . So far, G-quadruplex DNA structures have been created by telomere sequences of several species, and X-ray structure analysis and NMR structure analysis have been performed. The G-quadruplex DNA structure has been studied as a different field from the t-loop structure, which is considered to be another telomeric terminal structure. Moreover, since the human telomere sequence is a repeat sequence of TTAGGG and contains adenine (A), there have been few reports on this G-quadruplex DNA structure.

Last year, a paral lel G-quadruplex DNA structure was reported that was fundamentally different from the antiparallel G-quadruplex DNA structure already reported for the first time in human telomere sequences. This structure is a quadruplex crystal structure grown at a K ⁺ concentration close to the intracellular concentration using four consecutive human telomeric DNA repeat sequences, and Κ + is found in the crystal structure. In this molecule The folding and appearance of DNA in quadruplexes is fundamentally different from the antiparallel G-quadruplex DNA structure containing Na +. All four DNA strands are in a parallel relationship, and three trinucleotide loops bridging them are arranged like a propeller outside the quadruplex core. An adenine residue in each TTA-bridged trinucleotide loop (T-T-A loop) is inserted back between two residues of thymine. Such a DNA structure indicates that telomeres can be easily folded and unraveled. In the laboratory of the present inventors, it was observed by NMR that the parallel G-quadruplex DNA interacted with the telomeric protein, hTRF2-DBD. To be precise, this G-quadruplex DNA structure has the same ssDNA sequence as that reported in the paper: 5'-TAGGGTTAGGGT-3 '(SEQ ID NO: 18). Sequence: formed by 5'-TTAGGGTTAGGG-3 '(SEQ ID NO: 1), but since consecutive guanine bases are conserved and the same telomere sequence is used, G formed by this ssDNA -It is considered that the quadruplex DNA has almost the same structure as the paper. This sequence difference may be related to the susceptibility of the G-quadruplex DNA structure to be formed. From these considerations, in this study, we conducted an interaction experiment using ssDNA that forms G-quadruplex DNA that interacted with hTRF2-DBD. We are also conducting experiments on interaction by NMR, and the main focus of this research is the presence of G-quadruplex DNA related to the measurement of dsDNA interaction and t-loop structure formation and the function of telomere proteins. As can be discussed, we are trying to measure the interaction with MRF2-DBD with Biacore.

First, we attempted to form G-quadruplex DNA. Previous studies have shown that a monovalent metal cation is required to form G-quadruplex DNA. We started by examining what kind of properties this structure actually has. For the structure confirmation, a CD spectrum measured with a measuring instrument J-720 manufactured by JASCO Corporation was used. The spectrum obtained by circular dichroism (CD) spectroscopy is the most widely used G-quadruplex DNA structure for structural studies. This is due to the characteristics of G-quadruplex DNA. The structure of G-quadruplex DNA is broadly classified according to the combination of the orientations of the DNA strands formed by the four Rich strands (parts). The reason why the CD spectrum is often used is that the absorption wavelength of the CD generated at that time is characteristically observed in two patterns, whereby the structure can be easily confirmed. In the parallel structure, a CD spectrum having a positive maximum at 265 nm and a negative maximum at 245 nm was observed. In the structure, a CD spectrum with a positive maximum at 295 nm and a negative maximum at 265 nm is observed [Figure 10]. This property is mainly Teromea array of Saccharomyces Cerevis iae advanced the study (G -! _3), that by the Teromea array of Oxytrich (G ₄ T ₄₎ and Tetrahymena themophi Teromea array of la (G ₄ T ₂₎ G -Observed in quadruplex DNA. This has also been studied in the human telomere sequence (TTAGGG), which was confirmed in this study. Furthermore, in this study, we focused on ssDNA solubilized buffers to further clarify the conditions under which the CD spectrum had a specific absorption. The CD spectrum was also used for this measure. As a result, when only K + was present in the buffer, the G-quadruplex DNA formed a parallel structure, and a CD spectrum having a positive maximum at 265 nm and a negative maximum at 245 nm was observed. On the other hand, when Na + is present, the G-quadruplex DNA forms an antiparallel structure, and a CD spectrum with a positive maximum at 295 and a negative maximum at 265 nm is observed. I understand [Fig. 10]. This result was the same as the structure of human telomere sequence sstr24 + (5′-TTAGGGTTAGGGTTAGGGTTAGGG-3 ′) (SEQ ID NO: 4) reported in a previous study. However, there is one major difference from these comparisons. That is, the CD spectrum when annealing with K + only buffer is clearly different. In this study, we also performed G-quadruplex DNA annealing with sstr24 + to confirm this result, but the result was as reported. This suggests that sstr24 + cannot form a single stable G-quadruplex DNA in the presence of K +. Considering the absorption position of the CD spectrum, it is apparent that sstr24 + is a state in which antiparallel structure, parallel structure, and slight minor structure coexist under this condition. On the other hand, sstrl2 +, on the other hand, produced only a perfectly parallel structure under these conditions [Figure 10]. In this study, we investigated whether the stability of the G-quadruplex DNA formed depends on the length of the telomere DNA sequence. In this measurement, the antiparallel structure was determined and sstr22 + (5'-AGGGTTAGGGTTAGGGTTAGGG-3 ') (SEQ ID NO: 3) two bases less than sstr24 + and sstr26 + (5'-TTAGGGTTAGGGTTAGGGTTAGGGTT-3' ) (SEQ ID NO: 16) was used. As a result, sstr26 + showed a CD spectrum having the largest amount of parallel structures. Conversely, sstr22 + forms the most abundant anti-parallel structure upon annealing in the presence of Na +, and the structure seems to form a very stable structure. Therefore, the previously reported sstr22 + antiparallel G-quadruplex DNA may have been structurally analyzed by bandits R regardless of its length. To summarize these, G- formed by the length of telomere DNA The structure of the quadruplex DNA was found to be restricted. In other words, it was considered that the parallel structure was difficult to be formed depending on the length of the telomeric 7 DNA, and that once formed, the structure was easily dissociated and easily unraveled. On the other hand, it was found that the antiparallel structure is always easy to form and that the structure itself is stable. The antiparallel structure has been extensively studied in human telomeric DNA sequences, and its structure and properties are known. It is also argued that the antiparallel structure of sstr24 + formed in the presence of K + has a higher heat denaturation temperature and is more stable than that in the presence of Na +. In addition, in this study, the comparatively short telomeric sequences tr8 + (5'-TAGGGTTA-3 ') (SEQ ID NO: 13), 5, G-trl2 + (5, -GTTAGGGTTAGG-3') (SEQ ID NO: 1 4), trl3 + (5'-GTTAGGGTTAGGG-3 ') (SEQ ID NO: 2) and 5, C-trl3 + (5'-CTTAGGGTTAGGG-3') (SEQ ID NO: 15), and further on the 5 'side of trl3 + 5 'C-trl7 + (5'-CATCATTAGGGTTAGGG-3') (SEQ ID NO: 17) (SEQ ID NO: 17), which forms a G-quadruplex DNA with a nucleotide sequence that has no relation to the telomeric sequence CD measurement was performed, but almost the same spectrum was observed in the telomere sequence of this length.

Telomere DNA is in the nucleus, and in its environment there are naturally divalent metal cations in addition to Na + and K +. Research papers on this have been reported. The telomere sequence used was that of yeast, but it was reported that Ca ²⁺ and Mg ²⁺ stabilize parallel G-quadruplex DNA and revert from antiparallel to parallel. . Therefore, in this study, the buffer containing Ca ²⁺ and the buffer containing Mg ²⁺ were prepared and annealed. However, the human telomeric sequence did not show such noticeable CD spectral changes, but only destabilized the reverse-parallel G-quadruplex DNA structure.

NMR confirmed the interaction of MRF2-DBD with the sstrl2 + parallel G-quadruplex DNA structure [Figure 10]. However, this double-length sstr24 + was unable to form only a parallel structure of G-quadruplex DNA. Therefore, the concentration dependence of ssDNA during annealing was examined. The concentration of ssDNA is also important in annealing dsDNA. The darker the dsDNA is formed. Therefore, the condition was examined using sstr24 +, which is twice as long as sstr26 + and sstrl2 +, where the parallel structure was most confirmed. As a result, it was found that the higher the concentration, the easier it is to form a parallel structure. In particular, for sstr26 +, almost perfect parallel structure was confirmed at about 500 M at ssDNA concentration. However, with sstr24 +, only perfect parallel structures 31 did not form. Therefore, in the measurement of the interaction with Biacore in this study, the antiparallel G- Heavy helical DNA was used. For sstrl2 +, for which an interaction was observed by NMR, only a parallel structure can be formed, and the interaction was measured using this.

 In the Biacore assay, there have been some refinements and considerable time has been spent deciding how to measure the G-quadruplex DNA-telomere protein interaction. The reason is that hTRFl-DBD and TF2-DBD were used to interact with G-quadruplex DNA, but the interaction as obtained by NMR could not be measured. Interaction with hTRF DBD resulted in aggregation that could not be measured in Biacore, and RF2-DBD could not be confirmed because the interaction was too weak. G-quadruplex helix DNA was biotinylated and immobilized on the SA chip.However, this is the only method that consists of biotinylated ssDNA alone, so it is not clear whether G-quadruplex helix DNA forms a parallel structure. Yes, and suggested that the portion recognized by the protein may be impaired by immobilization. Therefore, we considered fixing telomere protein as a countermeasure.

 The method used in this study created a His-tag fusion protein expression system, purified it, and immobilized the protein via Ni using an NTA chip. The construction of this expression system was relatively smooth. However, there was one problem during the experiment, and the concentration of hTRF2-DBD after restriction enzyme treatment could not be recovered as expected. Therefore, primers were purchased and amplified by the PCR method. As a result, the RF2-DBD portion could be recovered, and the His-tag-fused hTRF2-DBD expression system could be easily constructed.

 After creating the His-tag fusion hTRF2-DBD expression system, it was actually expressed and purified. As a result, the N-terminal His-tag fusion protein was higher in both expression level and recovery [Figure 9]. For purification, cation exchange chromatography and gel filtration chromatography were mainly used. In column purification using cellulose phosphate, a cation exchange resin, the elution salt concentration was determined using a batch method in which the resin and protein solution were mixed and the target protein was eluted. The concentration was 300 mM or more for N-terminal His-tag and 400 mM or more for C-terminal His-tag. As a result, most of the contaminants were removed in the same manner as in normal hTRF2-DBD purification. Finally, this was concentrated and purified with a gel filtration column to obtain a final sample. A part of this was exchanged for running buffer for Biacore and used for immobilization.

The results of the interaction with Biacore are discussed. As a result of the interaction measurement using the Biacore device, in the ss trl2 +, the interaction with the parallel structure is more significant than the interaction with the antiparallel structure. It was confirmed that it was about two orders of magnitude stronger [Tables 3 and 4]. For sstr24 +, it was suggested that the antiparallel structure and hTRFl-DBD interact more specifically than MRF2-DBD. As described above, it was confirmed that sstrl2 + and hTRF2-DBD interacted with Biacore as in the interaction experiment with NMR. At the same time, based on the result of interaction with the parallel G-quadruplex DNA structure of sstr8 +, when the human telomere sequence forms a G-quadruplex DNA with ssDNA, the structure is a trinucleotide loop. -Predicted the possibility of interacting with A loop. However, in that case, G-quadruplex DNA formed by long ssDNA (tr22 +, tr24 +) also interacts with TTA-loop while being antiparallel. However, as a result, sstr24 + was more likely to interact with telomere proteins. The reason for this is that hTRF2-DBD does not interact, whereas sstr24 + is unstable because sstr22 + has a more stable antiparallel G-quadruplex DNA structure than sstr24 +. It is considered that the differences in the interaction with hTRF2_DBD did not appear depending on the length and the length of some sequences. In other words, it is thought that the antiparallel structure was dissociated because of the unstable structure, and a structure that was convenient for the RF2-DBD coupling was formed. However, from the confirmation of the G-quadruplex DNA structure by CD spectrum and the strength of the interaction by Biacore, it seems difficult to elucidate the mechanism of the interaction in detail.

 What we can certainly say in this study is that the parallel G-quadruplex DNA formed by sstrl2 + interacts with hTRF2-DBD more strongly than its antiparallel G-quadruplex DNA. In addition, it was confirmed that the antiparallel G-quadruplex DNA of sstr24 + interacted with hTRF2-DBD, which was also found to interact with hTRF1-DBD. In the future, we would like to create an expression system for His-tag fusion hTRFl-DBD and confirm its interaction with G-quadruplex DNA on Biacore.

[Example 3] Analysis of complex of telomeric protein TRF2 and DNA by NMR

Preparation of sample for R measurement

3.1 DNA preparation

Of non-labeled DNA is Yore ¹³ C / ¹⁵ N labeling of DNA which have been purchased from the Corporation BEX used was purchased from Nippon Sanso Co., Ltd.. In addition to trl2 +, the DNA used in the experiment was, as a comparative sample, the 8-mer sequence of TAGGGTTA (SEQ ID NO: 13) (hereinafter tr8 +), CCCTAACCCTAA (SEQ ID NO: The 12-mer sequence of 19) (a sequence complementary to trl2 +; hereinafter trl2-) was used. In addition, in order to examine the dependence of the Parallel structure on K ⁺ , samples in a buffer solution using Na + were prepared by changing the buffer conditions of trl2 +.

<< Annealing >>

Each DNA, which was lyophilized and sent in a single-stranded state, was dissolved in 50 mM KPB (pH 6.8) and 20 mM 75 mM KC1 solution, then placed in a microtube, covered with parafilm, and completely sealed. . Here, the freeze-dried DNA was dissolved in 50 mM NaPB (pH 6.8) and 75 niM NaCl solution in 20 O in the sample in the buffer using Na + to examine the K + dependency. To anneal them after the heating of 9 0 ^D C, 10 min floated in a constant temperature bath (SIBATA Co. BUCHI461Water Bath), as the temperature is room temperature (about 25 ° off the power to the thermostatic dregs C). After heating for 65 or 20 minutes, the thermostat was turned off and left as it was until the temperature dropped to room temperature.

<Buffer exchange>

 To completely replace the solvent contained in the purchased DNA with the target solvent (50 mM KPB (pH 6.8), 75 mM KC1 solution, or 50 mM NaPB (pH 6.8), 75 mM NaCl solution) Buffer exchange was performed for each buffer using.

《PH adjustment》

 The pH of the sample was adjusted to H.800 with NaOH and HC1 using a pH meter (PHM93, manufactured by RADIOMETER) for NMR measurement.

《Concentration test》

 The absorbance of the sample was measured using UV. Absorbance was measured at a wavelength of 260 nm. Here, since G-Quadrupl ex forms a special structure, 8 M Urea was added to the sample for concentration test, and the sample was completely boiled by boiling to conduct the concentration test. In addition, since the telomere sequence is TG-rich, an error may occur with a normal extinction coefficient. Therefore, the oligoparameter calculated by the sequence was used (see: ttp: // genset. bioweb. ne. jp /). The concentration of the sample was determined by substituting into the following equation.

 <trl2 +>

 DNA concentration.

= Absorbance of 260 belly X dilution (times) X single strand coefficient / molecular weight (single strand) The extinction coefficient is 29.7 and the molecular weight is 376.5.

 By calculation, the concentration of DNA was approximately 5.05 mM. '

 <tr8 +>

 DNA concentration

 = Absorbance at 260nm x Dilution (times) X Coefficient of single strand / Molecular weight (Single strand)

 The extinction coefficient is 28.48 and the molecular weight is 2464.7.

 By calculation, the concentration of DNA was about 9.7 mM.

 <trl2->

 DNA concentration

 = Absorbance at 260nm x Dilution (times) X Coefficient of single strand / Molecular weight (Single strand)

 The extinction coefficient is 31.83 and the molecular weight is 3534.36.

 By calculation, the concentration of DNA was approximately 7.9 mM.

3.2 Preparation of protein

Unlabeled · ¹⁵ N-labeled hTRF2 DNA binding domain (hereinafter DBD) was used as prepared in Example 1. Unlabeled and unlabeled RFD DBDs used in the comparison of interaction were also prepared in Example 1.

<< buffer exchange >>

 Buffer exchange to each buffer using a microdialyzer to exchange each protein for the target solvent (50 mM KPB (pH 6.8), 75 mM KC1 solution or 50 mM NaPB (pH 6.8), 75 mM NaCl solution) Was done.

《PH adjustment》

 The pH of the sample was adjusted to pH 6.800 with NaOH and HC1 using a pH meter (PHM93, manufactured by RADIOMETER) for NMR measurement.

《Concentration test》

 The absorbance of the sample was measured using UV. Absorbance was measured at a wavelength of 280 nm. The concentration of the sample was determined by substituting into the following equation.

 Sample concentration = absorbance at 280 nm X dilution (times) / molecular extinction coefficient

Since both hTRFl-DBD and hTRF2-DBD have four Trps and two Tyrs, the molecular extinction coefficient is 24.68. By calculation, the concentration of unlabeled hTRFl-DBD is about 0.8 mM, about ¹⁵ N-labeled hTRFl-DBD is about 1.7, about unlabeled MRF2-DBD is about 4.8, and about ¹⁵ N-labeled hTRF2-DBD is 7.OmM Met.

3.3 Preparation of complex sample

As a sample used for NMR measurement of the complex of hTRF2-DBD and trl2 +, a mixture of hTRF2-DBD and trl2 + at a concentration of 1: 1 was used. In the titration experiments, both the method of gradually adding DNA to protein and the method of gradually adding protein to DNA were used.

Circular dichroism (C D) measurement

In order to examine the structure of each DNA, measurement using CD was performed.

The principle of CD>

 Linearly polarized light consists of left circularly polarized light and right circularly polarized light. Circular dichroism (CD) is a phenomenon that occurs when left and right circularly polarized light interact differently with optically active molecules. The structure of DNA has several electronic transitions in the far ultraviolet wavelength region from 200 nM to 320 nm, which vary depending on the state of the DNA. As a result, the CD spectrum observed in this wavelength range differs depending on the structure of single-stranded DNA, double-stranded DNA, and G-quadruplex. In particular, CD is frequently used to study the secondary structure of proteins and nucleic acids because of its sensitivity in distinguishing different primary structures.

 The advantages of secondary structure studies with CDs are their ease and the ability to measure at low concentrations. The measurement is performed with a solution having a concentration similar to or lower than that of a normal UV absorption spectrum, and the measurement time is relatively short. Since the CD measurement refers to the average properties of the entire molecule, its accuracy is not as good as X-ray structure analysis or NMR measurement spectroscopy, but its characteristics have established a firm position in the three-dimensional structure studies of proteins and nucleic acids. are doing.

 The waveforms that have been established as characteristic of the G-Quartet structure to date are that the Ant i-Paral le l structure has a positive maximum at 295 nm, the waveform has a negative maximum at 265 nm, and the Paral le i structure The waveform has a positive maximum at 265 nm and a negative maximum at 245ηπι.

 The sample used for the measurement is

 1.tr l2 + (50mM KPB (pH6.8), 75mM KC1)

 2 tr l2 + (50inM NaPB (pH6.8), 75mM Ml)

 3.t r8 + (50mM KPB (pH6.8), 75ni KC1)

4.tr l2- (5 (kM KPB (pH 6.8), 75 mM KC1) For sample 1, samples before and after annealing were prepared to examine the difference in waveform before and after annealing. Samples 2 to 4 after annealing were used.

 The measurement was performed using a measuring instrument manufactured by JASCO Corporation; Model ί-720, and the attached standard measurement program was used. Measurement conditions were a square quartz cell (1 band), measurement temperature 293K, measurement wavelength 220-320nm, resolution 0. lnni, wavelength sweep speed 20mn / min, number of integrations 4 times, response lsec, band width 1.0 bold. The sensitivity was set to lOmdeg.

丽 R measurement

 3.4 NMR measurement of tΓl2 +

《2D 丽 R measurement》

NMR measurements were performed for structural analysis of trl2 + alone.

<Measurement conditions>

Using sample, and non-labeled trl2 +, ¹³ C / ¹⁵ N labeled TRL2 +

 Spectrometer '' Brueker AVA CE-500MHz (cryo-probe)

 AVANCE-800MHz manufactured by Brueker

The solvent · · · 50mM KPB (pH6. 8), 75ni KC1 (10% D 2 0, in 90¾ H ₂ 0)

5 (M KPB (pH6. 8 ), 75mM KC1 ( in 100% D ₂ 0)

※ 100% sample in D ₂ 0 was used to erase the signal of ¾0 become noise.

 Sample concentration '500 · ~ 1πιΜ

 Temperature 293Κ

Measurement2D 'H / ¹³ C CT-HSQC

 2D HCCH-C0SY

 2D HCCCH-C0SY

 2D HCCCCH-C0SY

 2D HCCCCCH-C0SY

 2D HCN

 2D HCNCH

 2D N0ESY

3.5 NMR measurement of complex of trl2 + and hTRF2-DBD 《Titration experiment》

 Titration experiments were performed to observe changes in the signals of MRF2-DBD alone and the hTRF2-DBD / DNA complex.

Measuring conditions>

Samples used unlabeled trl2 +, unlabeled tr8 +, unlabeled trl2-unlabeled hTRF2_DBD, ¹⁵ N-labeled M F2-DBD

Unlabeled RF 1_DBD, ¹⁵ N-labeled HTRF 1-DBD

 Spectrometer '· Brueker AVANCE- 500MHz (cryo-probe)

The solvent · · · 50mM KPB (pH6.8) , 75mM Cl (10% D 2 0, in 90% H ₂ 0)

50mM NaPB (pH6.8), 75mM NaCl (10% D 2 0, in 90% H ₂ 0)

 Sample concentration: 100 M to 200 M

 Mixing ratio, · 'Protein: DNA = 1: 0, 1: 0.25, 1: 0.5, 1: 0.75, 1: 1, 1: 1.25, 1: 1.5,

 1: 1.75, 1: 2

 DNA: protein = 1: 0, 1: 0.25, 1: 0.5, 1: 0.75, 1: 1, 1: 1.25, 1: 1.5,

 1: 1.75, 1: 2

* In the two-dimensional measurement, the unlabeled protein is mixed with the unlabeled DNA at a constant DNA concentration to observe the change in the DNA signal, and in the one-dimensional measurement, the protein concentration is constant to observe the protein signal change. The experiment was performed by mixing unlabeled DNA with ¹⁵ N-labeled protein. Temperature 293K

 MeasurementID 1H

2D ^] H / ¹⁵ N HSQC

<NMR measurement for structural analysis>

NMR measurements were performed to determine the interaction site of the hTRF2-DBD-DNA complex.

<Measurement conditions>

Using sample · · · ¹³ C / ¹⁵ N labeled TRL2 +, unlabeled HTRF2- DBD

 'Brueker AVANCE_500MHz (cryo-probe)

The solvent · · · 50mM KPB (pH6.8) , 75mM KCl (10% D 2 0, in 90% H ₂ 0) 50謹KPB (pH6.8), 75mM KC1 (in 100% D ₂ 0)

 Sample concentration · · '500 M

 -Rl2l: TRF2-DBD = l: K 1: 2

 * The mixing ratio in this experiment was trl2 +: hTRF2-DBD = 1: 1, where no change was observed from the results of the titration experiment, since it was not clear at what ratio trl2 + formed a complex. Next, measurement was performed at a ratio of 1: 2 so that all trl2 + became a complex.

 Temperature 293K

Measurement2D ¾ / ¹³ C CT-HSQC

2D ^! H / ¹⁵ N HSQC

DNA structural analysis

 Analysis of tr 12 + alone has not been completed yet, but NMR studies show that KC 1 has a quadruple parallel structure consisting of dimers. We briefly describe the DNA analysis method used in this study.

《Classification of W ¹³ C CT-HSQC, HC _n CH-COSY signal of DNA residue》

First, we classified the signals, that is, which signal belongs to which residue (however, it is unknown which signal is in which residue). ^! H- ¹³ C CT- HSQC H-side 1-7 ppm and C-side 35-90 ppm, a signal of the DNA's own residue was observed. For HCCH-C0SY, HCCCH-COSY, HCCCCH-COSY and HCCCCCH-COSY, the correlation with any measurement is observed, so the signal of the same base should be the same ppm. Therefore, the bases could be classified from the H m. Furthermore, HCCH- the COZY ¾ ,, ₂ .. C signals can be observed in, "¾ of Ή / CT- HSQC by comparing the range of., _2" ,, ₂ WC CT-HSQC of signals Classification has come out. HCCCH-C0SY, HCCCCH-COSY, and HCCCCCH-COSY could be classified in the same way.

 《Classification of N / 'CT-HSQC signal of HCNCH, HCN, base》

The signal was classified for HCNCH. Since this measurement can observations correlation with 6 / 8H bases, it was possible to classify the signal as compared to the ^{'H / 13 C CT-HSQC} .

Next, HCN signals were classified. The HCN signal on the / 3-D-report side shows a correlation between Η _Γ and 1 / 9N of the base, and the signal can be classified by comparing with 'H / ¹³ C CT-HSQC. Was. In the base side HCN signal, a correlation of 6 / 8H of the base and 1 / 9N of the base was observed, and the signal was classified by comparing with 1 / 9N of the base of the HCN signal on the 3_D-report side. Was completed.

In a further ¹ H / ¹³ C CT- HSQC correlation of 6 / 8H and C bases it is observed, HCNCH if Ku was able to classify the signal by comparing the 6 / 8H of HCN base side . <Classification and attribution of own residue N0E>

In the N0ESY signal, the range in which N0E with the DNA's own residue is observed in 1.0 to 6.5 ppm, and the range in which N0E with 6 / 8H of the base is observed in 6.5 to 8.4 ppm. Therefore, by assigning N0E in the range of 6.5 to 8.4 ppm in the X-axis direction and 1.0 to 6.5 ppm in the y-axis direction, 6 / 8H of the base and N0E of the DNA self-residue should be clarified. I made it. First, the 6 / 8H (X-axis side) bases were classified by comparing / 8 / CT-HSQC, where the correlation between 6 / 8H and C was observed, and then by 分類 / CT-HSQC of the DNA's own residue. Eta _gamma of the self _{residues, Η 2 ,, 2 .., ¾} ,, Η 4., was classified in comparison with Eta ₅ ,,. of Eta signal. Once most of the signals could be classified, structurally similar residues were determined by identifying which residues were observed other than the own residue. As a result, most of the signals could be identified, and it was possible to identify which signal belongs to which residue by performing sequential assignment. Furthermore, residue assignment could be performed for 'H / ¹³ C CT-HSQC HCCH-COSY, HCCCH-COSY, HCGCCH-COSY, HCCCCCH-COSY. In addition, the assignment was performed for CH ₃ on the thymine residue / CT-HSQC.

《Chemical shift table》

 From the analysis results described above, a chemical shift table for the observed signals was created (Table 5). Result

 3.6 CD measurement.

 <Comparison before and after trl2 + annealing>

Comparing the CD spectra before and after annealing of trl2 +, the maximum before annealing was around 275 nm, the minimum was around 250 nm, the maximum after annealing was around 265 nm, and the minimum was around 245 nm. . From this, the structure of trl2 + before and after annealing is clearly different, and the CD spectrum of trl2 + after annealing is G-uadru lex showed a characteristic waveform. In other words, it was suggested that tr 12+ forms a parallel G-quadruplex structure.

《Comparison of trl2 +, trl2-, tr8 +》

 Comparing the CD spectra of trl2 +, trl2- and tr8 + (all after annealing), the maximum of trl2 + is around 265 nm, the minimum is around 245 nm, the maximum of tr12- is around 275 nm, the minimum is around 250 nm, and the maximum of tr8 + is 265 The area around the gall and the minimum were around 245 thighs. From this, the CD spectra of trl2 + and tr8 + showed waveforms characteristic of Paralel G-Quadruplex, but trl2- showed different waveforms. In other words, it was suggested that trl2 + and tr8 + form a Paralel G_Quadruplex structure, but trl2- is a different structure from trl2 + and tr8 +.

《Kl + Na + dependence of trl2 +》

Comparing the CD spectra of trl2 + in the presence of K + and trl2 + in the presence of Na ⁺ (all after annealing), the maximum of trl2 + in the presence of K + is around 265 nm, the minimum is around 245 nm, and the maximum of trl2 + in the presence of Na + Was at 295 nm and around 250 belly, and the minimum was around 265 nm. From this, the CD spectrum of trl2 + in the presence of K + shows a waveform characteristic of Paralel G-Quadruplex, and the CD spectrum of trl2 + in the presence of Na + shows the waveform characteristic of Ant i-Parallel G-auadruplex. showed that. Although no data is shown here, the buffer containing K and Na also showed the characteristic waveform of Paralel G-auadruple. In other words, it was suggested that trl2 + in the presence of K + forms a Paral lel G-auadruple structure, and that trl2 + forms an Ant i-Parallel G-Quadruplex structure in the presence of only Na ⁺ . Hereinafter, trl2 + forming the Paral lel G-Quadruplex structure is referred to as trl2 +, and trl2 + forming the Ant i-Paral lel G-Quadrulex structure is referred to as trl2 + (ant i).

3.7 NMR measurement of 7 tr 12 + Structural analysis

Results of NMR measurement of trl2 + • The residue assignments are as shown in Table 5. Table 5

 trl2 + chemical shift table

3.8 NMR measurement of hTRF-DBD and DNA

 << 1D NMR measurement of trl2- trl2 + trl2 + (ant i), tr8 + >>

Comparing the one-dimensional spectra of trl2- trl2 trl2 + (ant i) and tr8 +, the trl2- spectrum is a characteristically sharp signal when unstructured, and No signal around 10 12 ppm characteristic of imino protons was observed. On the other hand, the spectra of trl2 + trl2 + (ant i) and tr8 + were characteristically broad signals when the structure was adopted, and the imino proton of guanine was also observed. But imino There is a difference between these three signals in terms of the proton signal.In trl2 +, the middle of the three large signals is split into two, while in tr8 +, the middle signal is not separated and trl2 + In (ant i), several other minor signals were observed (Fig. 11). Therefore, it was suggested that trl2_, trl2 +, trl2 + (anti), and tr8 + form different structures.

<DNA: hTRF-DBD titration experiment>

 <tr l2: One-dimensional spectrum of TRFl-DBD and trl2 +: hTRF2-DBD>

 In the titration experiment of trl2 +: hTRF2-DBD, it was observed that the spectrum derived from DNA changed greatly as RF2-DBD was added to DNA, and the spectrum was separated. On the other hand, in the trl2 +: hTRFl-DBD titration experiment, as hTRFl-DBD was added to DNA, the signal broadened as a whole, and eventually the sample was denatured and became cloudy in the sample tube. This was visually confirmed, and the signal derived from DNA could not be confirmed in the NMR signal (Fig. 12). This is probably due to non-specific binding. These results suggest that mixing trl 2+ and RF2-DBD causes some structural changes, but mixing tr 12+ and hTRF 1-DBD does not result in specific binding.

<hTRFl-DBD: trl2 + 2D spectrum>

 When trl2 + was added to hTRFl-DBD, the signals of K374, Ε386, and the sc (side chain) disappeared (Fig. 13). However, the sample in the sample tube was confirmed to be denatured as trl2 + was added, and it is unclear whether this signal change was due to specific binding.

HTRF2- DBD: trl2 + 2D spectrum>

 When trl2 + is added to MRF2-DBD, the consecutive amino acid signals of Y462, Q463, Y465, G466, G468, W470, A471, A472, 1473, S474, K475, and N476 disappear or shift. (Fig. 14). This suggests that mixing RF2-DBD with trl2 + may cause some structural change in hTRF2-DBD from 462th valine to 476th asparagine.

<MRF2- DBD: 2D spectrum of tr8 +>

hTRF2-DBD to be carried out by the addition of tr8 + changes in the signal was observed _d

HTRF2- DBD: trl2- 2D spectrum> No signal change was observed even when trl2- was added to MF2-DBD.

<2D spectrum of hTRF2-DBD: trl2l (anti)>

 No signal change was observed even when trl2 + (anti) was added to MRF2-DBD. 3.9 NMR measurement of complex of hTRF2-DBD and tr 12+

TRL2 + and RF2- DBD complex of DNA side signal (base 6 / 8H_6 / 8C, 3- D- ribose of Η, ·, Ή / CT- HSQC signal CH ₃ thymine) Looking at, for Guanin It was confirmed that the signal was lost and shifted, and that the C の of thymine was also shifted overall, but that the seventh thymine was shifted significantly. This was the same for trl2 +: MRF2-DBD in the ratio of 1: 1 and 1: 2. Comparing the signals at trl2 +: RF2- DBD = 1: 1 and 1: 2, the signal signal at 1: 2 was slightly less, but the minor signal decreased. These results suggest that mixing trl2 + and RF2-DBD causes structural changes in tr12 + guanine and seventh thymine. However, the number of minor signals is so large that further measurement conditions need to be considered. Consideration

In this study, tr 12+ forms a Parallel G-auadrupklex structure in the presence of K ^+, suggesting that this structure interacts specifically with RF2-DBD. This is an interaction that is not seen in trl2 + (anti), tr8 +, and trl2-, and is thought to be due to the characteristic structure of trl2 + that forms the Parallel G-Quadruplex structure. In a G-Quadruplex in which there are many structural types, most are Anti-Parallel G-Quadruplexes.To form a Parallel G-Qiiadruplex, one structure consisting of two DNAs and four There are only two types of one structure consisting of DNA (see Figure 5). The trl2 + used in this study was not shown in the data, but the results of ultracentrifugation suggested that it was probably formed from two pieces of DNA. In a paper published in Nature in 2002, it was shown that a 12-mer DNA of TAGGGTTAGGGT (SEQ ID NO: 18) forms a dimeric Paral lei G_Quadruplex that forms a TTA loop. The results show that the current N0E analysis also predicts a TTA loop. These results suggest that trl2 + forms a Parallel G-Quadruplex in the dimer, and this structure has some interaction with RF2-DBD. From the results of the titration experiments, the interaction site of hTRF2_DBD was V462-N476 around the second helix. I was able to identify. This was mapped to confirm the site in the protein structure. The structure of F2-DBD is currently being analyzed, and it is known that the structures of DBD and hTRF DBD are almost the same.Therefore, mapping was performed by replacing the amino acid on MRF2-DBD with WRF DBD. (Figure 15). Although the site of the trl2 + interaction is currently being determined, it is likely that guanine and the 7th thymine are involved, and further measurements are needed in the future.

 Also, hTRF DBJ) and hTRF2-DBD have very high homology, and although their structures are almost the same, only hTRF2-DBD is known to interact with trl2 +. Although this is not clear, we believe that it may be due to slight differences in amino acid sequence and structure.

 It is unknown at present whether the G-quadruplex exists in the body or what role it plays. However, studies using G-QMdruplex have been conducted, and recent papers indicate that the development of HIV inhibitors is progressing. In this study, we are conducting structural analysis of the complex of G-Quadrupl ex and protein, but we plan to elucidate a more detailed three-dimensional structure. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial applicability

 According to the present invention, a G-quadruplex DNA capable of forming a complex with a telomere protein (TRF1, TRF2) has been provided. Further, according to the present invention, a complex of a telomere protein (TRF1, TRF2) and a G-quadruplex DNA is provided. Since telomeres are thought to be involved in the control of aging and canceration, use the DNA and complex of the present invention to analyze the mechanisms of aging and canceration and to design drugs such as anticancer drugs. Is possible. Sequence listing free text

SEQ ID NO: 1 shows the nucleotide sequence of trl2 +, SEQ ID NO: 2 shows the nucleotide sequence of tr13 +.

SEQ ID NO: 3 shows the nucleotide sequence of tr22 +.

SEQ ID NO: 4 shows the nucleotide sequence of tr24 +.

SEQ ID NO: 5 shows the amino acid sequence of TRF1-DBD.

SEQ ID NO: 6 shows the full length amino acid sequence of TRF1.

SEQ ID NO: 7 shows the amino acid sequence of TRF2-DBD.

SEQ ID NO: 8 shows the full length amino acid sequence of TRF2.

SEQ ID NO: 9 shows the nucleotide sequence of 5 'primer for N-terminal His-tag. SEQ ID NO: 10 shows the nucleotide sequence of a 3 ′ primer for N-terminal His-tag. SEQ ID NO: 11 shows the base sequence of the 5 'primer for C-terminal His-tag. SEQ ID NO: 12 shows the nucleotide sequence of the 3 ′ primer for C-terminal His-tag. SEQ ID NO: 13 shows the nucleotide sequence of tr8 +.

SEQ ID NO: 14 shows the nucleotide sequence of 5′-G-trl2 +.

SEQ ID NO: 15 shows the nucleotide sequence of 5, -C-trl3 +.

SEQ ID NO: 16 shows the nucleotide sequence of tr26 +.

SEQ ID NO: 17 shows the nucleotide sequence of 5, -C-trl7 +.

SEQ ID NO: 18 shows the nucleotide sequence of ssDNA reported in Nature's paper. SEQ ID NO: 19 shows the nucleotide sequence of trl2-.

Claims

1. in the presence of a cation,

 A telomeric protein of either TRF 1 or TRF 2 or a telomeric protein fragment comprising the DNA binding region thereof;

Guanine—a complex formed with DNA that has a quadruple helix structure.

 2. The complex according to claim 1, wherein the cation is a sodium ion or a potassium ion. Contract

 3. The complex according to claim 1, wherein the telomere protein or a telomere protein fragment containing the DNA binding region thereof is selected from the group consisting of the following proteins (A) to (D) and protein fragments.

 (A) TRF box 1 represented by the amino acid sequence of SEQ ID NO: 6

 (B) TRF 2 represented by the amino acid sequence of SEQ ID NO: 8.

 (C) a TRF1 fragment containing the DNA binding region of TRF1, represented by the amino acid sequence of SEQ ID NO: 5 (however, the first methionine residue may be deleted)

 (D) a TRF2 fragment containing a DNA binding region of TRF2 represented by the amino acid sequence of SEQ ID NO: 7 (the first methionine residue may be deleted)

 4. The complex according to any one of claims 1 to 3, wherein the DNA having a guanine-quadruplex structure has any one of the nucleotide sequences of SEQ ID NOs: 1 to 4.

 5. The telomere protein or a telomere protein fragment containing the DNA binding region thereof is TRF1 or a TRF1 fragment containing the DNA binding region thereof, and guanine—DNA having a quadruplex structure is an antiparallel guanine— The complex according to any one of claims 1 to 4, which has a quadruple helix structure.

 6. The telomere protein or a telomere protein fragment containing the DNA binding region thereof is TRF2 or a TRF2 fragment containing the DNA binding region thereof, and guanine—a DNA having a quadruple helix structure is composed of parallel guanine-14. The complex according to any one of claims 1 to 4, which has a heavy helical structure.

7. The complex according to claim 1, wherein the complex is selected from the group consisting of the following complexes (i) to (iv). (i) represented by the amino acid sequence of SEQ ID NO: 5, including TRF 1 DNA-binding region A complex of a fragment and a DNA having the nucleotide sequence of SEQ ID NO: 4 and having a guanine quadruplex structure

 (ii) a TRF2 fragment containing the DNA binding region of TRF2 represented by the sequence consisting of the amino acid sequence of SEQ ID NO: 7, and a DNA having the nucleotide sequence of SEQ ID NO: 1 and having a guanine quadruplex structure Complex with

 (iii) A complex of a TRF2 fragment containing the DNA binding region of TRF2 represented by the amino acid sequence of SEQ ID NO: 7 and DNA having the nucleotide sequence of SEQ ID NO: 2 and having a guanine quadruplex structure

 (iv) A complex of a TRF2 fragment containing the DNA binding region of TRF2 represented by the amino acid sequence of SEQ ID NO: 7 and a DNA having the nucleotide sequence of SEQ ID NO: 3 and having a guanine quadruplex structure

 (iv) A complex of a TRF2 fragment containing the DNA binding region of TRF2 represented by the amino acid sequence of SEQ ID NO: 7 and a DNA having the nucleotide sequence of SEQ ID NO: 4 and having a guanine quadruplex structure

 8. DNA having a base sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 13, 14, 15 and 16, and capable of forming a guanine quadruplex structure.

 9. The DNA according to claim 8, wherein the DNA has a guanine quadruplex structure.

 10. The DNA of claim 9, wherein the guanine quadruplex is a parallel chain guanine-quadruplex.

 11. The DNA of claim 9, wherein the guanine quadruplex is an antiparallel guanine-quadruplex.