JPH06510901A - Universal site-specific nuclease - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Universal site-specific nuclease field of invention The present invention focuses on the field of fusion proteins and their use as site-specific nucleases. Regarding using. More specifically, the fusion protein of the invention comprises a ligand -Ligand consisting of exonuclease or ligand-endonuclease- It is a protein hybrid.

It acts by recognizing and binding to sequences ("arrays"). When you recognize it, Each enzyme is identified by its cleavage pattern.

Restriction enzymes are used to manipulate DNA to create new recombinant molecules. Although often used by operators, the site specificity of these enzymes This tends to limit the usefulness of genetic engineering tools. any desired recognition sequence Research efforts have been frustrated by the unavailability of endonucleases to cleave There are many. Furthermore, some similar RNA restriction enzymes cannot be isolated from nature. , has hindered structural and functional research into RN.

To address this problem, we have proposed In recent years, oligonucleotides carrying reactive groups have been developed to specifically react with Reotide has been developed. For example, some metal chelates F exemplified below e-EDT8 [Boutor i n, ^, S et al., FEBS LetL, 17 2: 43-46 (1984); Chu et al., Pr.

e, Natl ^cad, sci, LIsA 82: 963-967 (19 85); Dreyer, G. B., et al., Proc. NatlAAcad, Sc. i, LIsA 82: 968-972 (1985): Boidot-Fo rget, i et al + Co5ptes Rendue AcaпASci, 5e Cu-zenatroline [Chen et al., Proc, Natl, Acad, Sci , uSA 83: 7141-7151 (1986); 02-9706 (1 989); Francois, J, C, et al. + Proc, l1at1. ^ca d, sci, ll5A 86:97O2-9706 (1 989) ] ; and ] Fe-porphyrin Le Doan et al. + Nuclei c Ac1ds Res, 15: 8643-8659 (19g?); Le Doan et al., Nucleic Ac1ds Res, 15: 864-86 59 (19g?)]’, Narano ■ Staphylococcal nuclease and RNase-5 are linked to oligonucleotides. p 85: 1349-1353 (198g); Lee et al. + Bioche mistry 27: 3197-3203 (1988) g Praseu th et al., Biochemistry 27: 3031-3038 (198B )].

Corey et al. [Biochemis Lry 28: 8277-8286 (1 989)] developed a novel binding domain for the enzyme. Relatively non-specific phosphodi Oligonucleotide at a unique site of staphylococcal nuclease, an esterase By fusing these, we created [)\Ibri-2 Donuclease. child No-brid nucleases such as can selectively cleave single-stranded M13sp7 DNA under a wide range of conditions. Here comes [Le Doan et al. + Mucleic Ac4ds Re++, 15: 7749-7760 (1937); PraseuLh, D et al., Proc, N aLl, Acad, Sci, USA 85: 1349-1353 (1988 j; Lee.

B, L, et al., Biochew+1try 27: 3197-3203 ( 1988); Praseuth, D, et al. B: 3031-3038 (198B)].

Summary of the invention The present invention provides a site-specific method that can selectively cleave any nucleotide sequence. Concerning endonucleases. The site-specific endonuclease of the present invention is , is a fusion protein consisting of a hybrid of a binder and a nuclease.

Initially, the ligand-specific receptor of the ligand-endonuclease fusion protein bind (covalently or A preselected nucleotide sequence can be prepared according to the invention by non-covalent binding). can be selectively cut. Gantt-endonuclease fusion of the present invention The ligand component of the protein is determined based on the affinity between the ligand and the receptor. Binds to receptors within the leotide sequence. Thus, the nucleotide containing the receptor By contacting a sequence with a fusion protein of the invention, one or a large number of offspring can be selected. The selected sequence can be digested, thereby converting the original nucleotide sequence into the desired region. It can be fragmented at certain points.

The invention particularly relates to fusions between staphylococcal nuclease and streptavidin. do. Such fusion proteins allow streptavidin-biotin interaction. recognizes biotin as a receptor. This biotin is produced by this fusion protein. may be directly or indirectly linked to the nucleotide sequence to be cleaved.

Biotin can be added to oligonucleotides that are complementary to part of the nucleotide sequence. Preferably, it is attached to a macromolecule. Biotin-oligonucleotides are added to this By binding (hybridizing) to the leotide sequence, the staphylococcus of the present invention Fungal nuclease-streptavidin fusion proteins contain this nucleotide sequence. It is possible to cut in a targeted manner.

Furthermore, the present invention relates to site-specific exonucleases. In this aspect consists of a hybrid of the fusion protein Higant and an exonuclease.

By using this site-specific fusion protein according to the present invention, nucleic acid Allows for controlled exonuclease digestion of the otide termini. That is, stipulated Controlled array! - Can be removed (hydrolyzed) in the right way, shortened thread length The otide fragment can be i4.

In another embodiment of the invention, the nucleotide sequence containing the receptor is first determined by its corresponding nucleotide sequence. receptor bound to a nucleotide sequence - anti-receptor Les, get a cus. The antireceptor component of this complex is the ligand for the fusion protein. is recognized by the DNA or RNA substrate to be cleaved. Bind the hybrid nuclease.

The present invention also provides a method for binding receptors indirectly to DNA or RNA molecules. Digestion of DNA or RNA. That is, the sequence to be cut is Instead of directly binding the receptor to a macromolecule that has an affinity for the substrate, Join to child. In this embodiment, a macromolecular "bridge" between substrate and receptor is , to bind to a specific recognition sequence within a DNA or RNA substrate, or to bind to a specific recognition sequence within a DNA or RNA substrate. can be designed to be randomly combined.

Brief description of the drawing Figure 1 shows a vector expressing a nuclease-streptavidin fusion protein. FIG.

Figure 2 is cleaved by a nuclease-streptavidin fusion protein. It is a process diagram showing the production of DNA substrate from alAl.

Description of preferred cho-sama ! Hiroshi In the following discussion, we will broadly refer to a number of terms used in recombinant DNA technology. Make use of it. Greater clarity and consistency in the specification and claims, as well as the scope of each term. In order to get a better understanding, we provide the following definitions.

Fusion protein 'ii: As used herein, the term ``fusion protein J'' refers to a single two or more macromolecules operably linked to form a macromolecule of one of which is a nuclease). be “functionally connected” The term refers to whether the catalytic or structural activity of each macromolecule is retained throughout the fusion protein. 6 refers to the binding (covalent or non-covalent) of macromolecules in such a manner that Ru.

For example, a fusion protein consisting of a hybrid rad between a nuclease and a ligand The fusion protein contains nuclease activity and binding activity or these activities. They will probably retain at least some of their sexuality. One of the components of the fusion protein In addition to the nuclease as -C1, at least one ligand is included. moreover , one or more ligands and a nuclease. can also be prepared.

>v: As used herein, the term "ligand" refers to a receptor (an anti-ligand). means any macromolecule or part thereof that has an inherent affinity for . The only limitation according to the present invention is that this term "ligand" and DNA).

・As used herein, the term "affinity" refers to the relationship between a ligand and a receptor or an antibody. meant to refer to the natural biochemical, chemical, or physical attraction between receptors . This attractive force causes the ligand to coexist with its corresponding receptor or its corresponding anti-receptor. result in covalent or non-covalent binding. In addition, the receptor binds to its corresponding anti-receptor Has IO affinity.

Receptor (antiligand): As used herein, the term "receptor" refers to or can be prepared. Examples of receptors include biotin, avidin, and Leptavidin, antigen, antibody, lactin, sugar conjugate, enzyme, substrate, adjuvant factors, inhibitors, cations, anions, hydrophobic sites, hydrophobic groups, hormones, vectors, toxins, transport proteins, vitamins, amino acids, sugars, membranes, liposomes, etc. These include, but are not limited to. catalytically or biologically active acceptor The term body refers to a receptor that can recognize and bind to its corresponding ligand. means.

Antireceptors In some situations, antireceptors are receptors bound to nucleotide sequences. A double layer that binds to the container and then acts as a receptor for the ligand portion of the fusion protein. be able to perform its functions. i.e., by directly binding to each other through anti-receptors. It is possible to bind a ligand and a receptor that cannot be I can do it.

■・The term substrate as used herein refers to cleaved by the fusion protein of the present invention. or refers to the DNA or RNA molecule to be hydrolyzed.

Fragment: The term “fragment” refers to a complete macromolecule (ligand, receptor). body, anti-receptor, or nuclease). these huge The term "catalytically or biologically active fragment" of a molecule refers to the catalytic or biological activity possessed, in particular by the molecules of the invention; Flags that retain all or part of their activity to allow receptor binding or sequence cleavage means ment.

Controversy: The term macromolecule “mutant” refers to a natural molecule or its fragments. molecules with great similarity in structure and catalytic activity, or their flags. refers to a ligand, receptor, antireceptor, or nuclease that is not identical to It means that. The term "variant" is intended to include isoenzymes and molecules. means. Variants are not necessarily derived from natural molecules. That is, two If the molecules have similar structure and catalytic activity, the composition of one of these molecules or the secondary, tertiary or quaternary structure is not identical to that found in the other. Even when not, these molecules are referred to as mutants as this term is used herein. considered a body.

■, Catalytic activity of fusion protein The present invention relates to Site-specific, which can be cleaved at any or multiple selected sites It provides a unique endonuclease. Furthermore, the present invention provides DNA or R A defined number of nucleotides from a specific end of an NA molecule (single-stranded or double-stranded) Relating to site-specific exonucleases useful for selective digestion.

According to the invention, the site-specific exonuclease or endonuclease of the invention The nuclease is a fusion protein between the nuclease and the ligand. Created by creating quality. This fusion body constitutes the fusion tongue 7 The catalyst is prepared in such a way that the catalytic activity of each component is maintained. Generally, the fusion of the present invention Synthetic proteins function in a similar manner. That is, the ligand component of fused tanno(lithium) dictates the location where the other catalytic activities of this fusion protein will act. Immediately In other words, the binding activity of the ligand has an affinity for the "recognition sequence", and this recognition When the sequences are joined by the fusion protein, this fusion protein The enzyme moiety cleaves or hydrolyzes within or near this recognition sequence. Main departure A "recognition sequence" when used with a light fusion protein is one that is closely associated with a binding receptor. Defined as related specific sequences.゛ The mode of action of the cleavage by the endonuclease fusion tongue 1 of the present invention is limited to type 1. Although similar to the enzymatic function of endonucleases, one important difference is evident. It is. In the present invention, the ligand portion of the fusion protein binds to the recognition sequence. Recognizes the receptor and binds to it, causing nuclease and cleavage to occur Place the arrays very close together. In this way, it binds (covalently or covalently) to the receptor. Any recognition sequence that can bind to the fusion protein of the present invention (covalently or non-covalently) , resulting in cleavage within or near the particular recognition sequence.

It is not necessary to cleave any DNA or RNA molecule or sequence according to the present invention. Wear. This DNA or RNA substrate is the endpoint of the fusion protein to the substrate. It is only limited by the nuclease moiety. For example, the fusion tag of the present invention The protein is a double-stranded fusion protein of the present invention with a specific endonuclease. When used to create double-stranded DNA, it will cut the double-stranded DNA. Similarly (This invention To cleave single-stranded DNA or RNA according to The endonuclease portion is a single-stranded DNA or RNA-specific endonuclease. nuclease, such as staphylococcal nuclease.

Exonuclease digestion of single-stranded or double-stranded DNA or RNA molecules has been This is carried out using a known exonuclease. These exonucleases Although controlled digestion is possible, it is not common to obtain molecular populations of specific lengths. This is only possible depending on the method (i.e. enzyme concentration, time, temperature and/or or control hydrolysis by changing enzyme conditions). However, according to the present invention provides controlled, site-specific digestion of single- or double-stranded nucleic acid molecules. This is achieved by a method in which specific ends of a nucleic acid molecule are shortened to a predetermined length.

In one embodiment, a fusion protein of the invention binds to a substrate nucleotide sequence. A fusion protein is placed near the end of the substrate that specifically binds to the receptor and is to be hydrolyzed. protein exonuclease is attached. Fusion proteins to this region of the nucleic acid molecule Once the molecules have joined, the exonuclease begins to digest the specific ends. This fusion Digestion is complete when the protein is removed from its nucleic acid substrate.

In accordance with the present invention, receptors are shared with specific sequences (referred to herein as "recognition sequences"). Combine or non-covalently. Fusion proteins can be formed by either covalent or non-covalent bonding. by binding the ligand portion of the protein to the receptor-nucleic acid complex. , the exonuclease hydrolyzes the nucleic acid terminus located near the ligand binding be able to.

Exonuclease digestion to the extent of removing the ligand removes the enzyme from its substrate. It has the effect of eliminating exonuclease digestion. in this way, The positioning of receptors near the terminus determines the extent of digestion of a particular terminus.

That is, the fusion protein is linked to the nucleotide sequence by receptor-ligand interaction. and initiate hydrolysis of the specific terminus by exonuclease. acceptance When the nucleotides within the containing sequence are removed by hydrolysis, the fusion protein The quality is effectively dissociated from interacting with the nucleotide sequence, thereby making it possible to Inhibits hydrolysis of fixed ends.

To facilitate detachment of the fusion protein from interaction with the nucleotide sequence It may be preferable to use a continuous flow reaction rather than a batch reaction. batch anti In response, the fusion protein is activated by binding of the fusion protein to the receptor-substrate. non-specific hydrolysis or cleavage due to accidental contact instead. Can be done. A continuous flow system can be used with the exonuclease or endonuclease of the present invention. This system can be used to bind both fusion proteins of the present invention. Requires the use of columns, filters, gels, foams, etc. fusion protein By passing the substrate-receptor through a matrix containing the site-specific cutting or hydrolysis of the substance becomes possible. This system allows easy removal of digestion products Therefore, non-specific interactions are prevented.

The fusion protein may be non-covalently attached to a continuous flow matrix. However, it is preferred that the bond be covalently bonded to the matrix. fusion protein fusion protein in any way that does not destroy the desired biological and catalytic activity of the protein. can be immobilized (immobilization of fusion protein through receptor-ligand interaction) (including formulation). For example, a fusion protein may contain two Gantts to form a fusion protein. is attached and immobilized on a matrix, and then binds to the receptor bound to the substrate. can be done.

The reaction conditions (time, temperature, pH, and salting v7t) using the enzyme of the present invention are as follows: Nuclease and ligand components of the fusion protein and the site to be cleaved and the number of nucleotides to be hydrolyzed. usually , the conditions used are those that are optimal for the natural unfused nuclease.

Such conditions are well known in the art. A person skilled in the art would be able to change the conditions and Obtain optimal nuclease activity and optimal binding activity for fusion proteins of be able to. Respective fusions using well-known nuclease and affinity assays Optimal reaction conditions for a protein can be determined.

■ Types of fusion proteins The fusion protein of the present invention has two basic activities associated with these fusions: It has nuclease and ligand binding activities. This fusion protein is The protein is produced in a way that retains the respective activities of the macromolecular components that led to the synthesis of the protein. to be accomplished. That is, the fusion protein of the present invention consists of at least two types of macromolecules, One is related to nuclease activity and the other is related to the binding activity attached to it. have.

Those skilled in the art will be able to operably link multiple nuclease and/or receptor molecules. to create numerous fusion protein “seeds” with multiple activities and therefore multiple functions. I'm sure you understand that. For example, the fusion proteins of the invention Donuclease, exonuclease and two different ligand molecules (same binding to another receptor within the nucleic acid molecule). I can do it. When this multifunctional fusion protein is contacted with a receptor-containing substrate, specific Site-directed hydrolysis of the terminal and pre-selected sites within the substrate sequence or The result of nearby endonuclease cleavage will be obtained. nuclease, The combination of receptors and/or anti-receptors depends on the substrate used and the desired result. will be readily apparent to those skilled in the art.

A Nuclease At least one component of the fusion protein of the invention has nuclease activity.

Protein nucleases of the invention include exonucleases and This includes both endonucleases and endonucleases.

Any macromolecule (protein or otherwise) that exhibits nuclease activity is included in the present invention. These include metal chelates, non-protein enzymes (riboza catalytically active proteins (enzymes), and catalytically active proteins (enzymes). Not allowed. Such metal chelates include Fe-EDTASCu-zenatro phosphorus, Fe-porphyrin and Fe(If) bleomycin [Carter et al. , Proc, Natl, Acad, Sci.

11sA 87:9373-9377 (1990) and Sigman et al. Annu, Rev, Biochet 5'l: 207-Q36 (1 990)] will be included. Fusion of the invention using protein nucleases Preferably, the protein is made. However, one aspect of the invention is that non-tampering Good quality cheI! 1stry 25: 4478-4482 (1986): Been, M, D, et al. 5science 239: 1412|1416 ( 198 8), and Doudna et al., NaLure 339: 519-522 ( 1989)].

Exemplary exonucleases for making fusion proteins of the invention include RNA and exonucleases specific for both DNA, e.g. Ase 11 Exonuclease I11, Exonuclease Vll, Lambda Ex Sonuclease, Jaenia nuclease, nuclease Ba13 L nuclease pH ribonuclease B, cereus, ribonuclease CI-3 , ribonuclease H1 ribonuclease phy+, ribonuclease PhyM , ribonuclease Tl.

Includes ribonuclease T2, and ribonuclease U2.

The endonuclease used according to the invention may be any nuclease known to the person skilled in the art. May include Azera. It is thermostable and recognizes only 4 nucleotides. Preferred are endonucleases. Such endonucleases include Has Is7->fls0976)] is included. Creating the fusion protein of the present invention The only requirement is that the endonuclease used is a non-specific nuclease. that is, in its natural unfused state, the endonuclease This means that the enzyme does not recognize or bind to a specific recognition sequence.

The endonuclease fusion proteins of the invention have substrate specificity for the desired chain. an appropriate endonuclease with is included as a component of the fusion protein As long as double-stranded or single-stranded RNA and DNA molecules can be digested.

Fusion proteins for cleaving single-stranded DNA at specific sites according to the invention A preferred endonuclease component of is staphylococcal nuclease. single strand The RNA can be derived from the ribonuclease RNase-8, ribozyme, or Staphylococcus nuclease. Preferably, cleavage is performed by a fusion protein containing clease. double strand D NA is Staphylococcal nuclease, or Allul, Haell or Ta It is preferable to cleave with a fusion protein containing either q(α)l as a component. However, on the other hand, RNase A [Shalongo et al. Digest with fusion 8 protein of 23:4820-4825 (1989)] I prefer λ.

B Ligand The conjugate component of the fusion protein of the invention recognizes its corresponding receptor and Any macromolecule or part thereof that can bind to the LS. present invention The only restriction that follows is that if this fusion tannin is attached to component b (nucleic acid molecule RN This means that it cannot contain A or DNA. Table 1 (Up to this invention) Figure 2 illustrates possible ligand, receptor combinations for use with this lt+'. Like Specifically, the ligand portion of the Nogando-nuclease fusion has affinity for the receptor; It is a catalytically active protein. In this method, tanno(lithium) - protein/macroprotein fusions (i, obtained by recombinant DNA methods well known to those skilled in the art) This IJ Gantt is capable of producing fusion tangents by well-known chemical means. The nuclease part of the stomach.

Biotin: Avidin, streptavidin, etc. Avidin, streptavidin, etc. D: Biotin Antigen: Antibody Lectin: sugar consucinate Sugar Funshgate: Lectin Enzyme disubstrates, cofactors, inhibitors, etc. Cation: Anion Hydrophobic site - Hydrophobic group Receptors: transport proteins such as hormones, effectors, toxins, etc. Jlt: vitamins, Amino acids, sugars, etc. 115 and length: So---------get fusion protein fee. The catalytically active protein ligand used for this purpose has an affinity for the receptor. Contains all kinds of proteins. Such ligands include avidin, strep toavidin, antibodies or antigens, enzymes, lectins, hormones, effectors, or or toxins, but are not limited to these. The protein-transmitting ligand is an antibody. In some cases, either whole antibodies or Fab fragments are used to test their corresponding bind to antigen receptors. Alternatively, the antigen or its catalytically active fragment can be The component is the ligand portion of the fusion protein of the invention.

The most preferred protein ligands for use in accordance with the invention are avidin or protein ligands. leptavidin and these bind to its corresponding receptor biotin .

A preferred non-protein ligand component of the fusion protein is biotin or its like. A catalytically active variant or fragment of Biotin as a ligand When used as a nucleic acid, the receptor that binds to the nucleic acid is avidin or streptavidin. Isolation of each part of the fusion protein (i.e., nuclease and ligand) is independent. The components of the invention can then be chemically fused in vitro. Obtain the fusion protein. In this case, substantially pure natural or recombinant protein proteins or non-proteins are fused by well-known chemical means.

and separate. In this preferred embodiment, the protein-protein fusion molecule to functionally link multiple genes encoding ligands and nucleases. obtain by doing so. In order to combine these genes in this way, each gene must be The active site is linked in the proper reading frame at the DNA level. This allows the protein translated from the “hybrid” gene to such that the quality contains the activity or part of the activity of each gene.

A. Nuclease or ligand gene cloning using any of a variety of methods. Using nuclease or ligand genes for the creation of fusion proteins of the invention Cloning the child. One such method is to use nuclease or ligand For the presence of inserts containing genes, DNA inserts (nucleases or To analyze a shuttle vector library (derived from m cells expressing Gant). is necessary. Such an analysis involves transfecting cells with a vector and then carried out by assaying the expression of nuclease or ligand catalytic activity in can do. The preferred method for cloning these genes is It requires determining the amino acid sequence of the crease or ligand molecule. child To do this, the nuclease or ligand protein is purified and automatically sequenced. It can be analyzed with a decision machine. Alternatively, both molecules can be treated with anogen bromide or Fragmentation with proteases such as papain, chymotrypsin or trypsin [04ke, Y, et al., J. Biol, Chem, 2 57+9751-9758 (1982): Liu, C. et al., InLj, Pe. pt, Protein Res, 21: 209-215 (19W3)].

Although it is possible to determine the entire amino acid sequence of these proteins, Preferably, the peptide fragment is sequenced. Peptide is 10 amino acids When longer than the length of the gene, this sequence information generally or Gantt genes).

Once ■ or more suitable peptide fragments have been sequenced, these Test for DNA sequences that can encode. Since the genetic code is degenerate, more than 1 A specific amino acid can be encoded using a codon that occurs [11atso n, J, D,.

[Molecular biology of genes, 3rd edition + 1. A, Benja+ein, Inc. , + Menlo Park, C^ (1977), pp, 356-35.

Analyze these peptide fragments to determine which oligonucleotide has the lowest degree of degeneracy. Identify the amino acid sequence hooded by Otide. This results in a single codon This is done by identifying sequences containing amino acids that are only covered by preferable.

If such an amino acid sequence is only hooded by a single oligonucleotide, However, if the amino acid sequence is determined by any one set of similar oligonucleotides, There are many things that can be hooded. Importantly, all members of this group It contains an oligonucleotide that can encode a tide fragment and therefore contains the same nucleotide sequence as the gene encoding the peptide fragment of only one member of this set has the null of the gene. It contains the same nucleotide sequence as the nucleotide sequence. This configuration member is present in the group and the DNA and node are present even in the presence of other members of the group. Peptides can be hybridized using a single oligonucleotide. Unfractionated oligos were cloned using the same method used to clone genes encoding Any set of nucleotides can be used.

In exactly the same manner as described above, the peptide fragments can be hooded. An oligonucleotide having a nucleotide sequence that is complementary to a oligonucleotide sequence or set of sequences. Ligonucleotides (or sets of oligonucleotides) can be used.

Suitable oligos capable of encoding fragments of nuclease or ligand genes a set of nucleotides or oligonucleotides (or is an oligonucleotide or oligonucleotide complementary to the oligonucleotide set. identification (using the method described above), synthesis, and dependence on the source of the gene. DNA or cDNA. Make it loud. Isolation of eukaryotic genes is usually accomplished by screening eDNA libraries. DNA library is used to isolate prokaryotic genes. Use brary. The method of hybridizing nucleic acids is described by Maniati S, T, et al. [[Molecular Cloning, Laboratory Manual], 2nd edition, Cold spring Harbor, NY (1989)] and llaymes, B, D, et al. [[Nucleic Acid I\Ibridization, Practical Approach].

Disclosed by IRL f’ress, 1ashingLon, DC (1935) (These documents constitute a part of this specification). Preferably, the source of DNA or cDNA is enriched with the desired sequences. This way Enrichment was achieved by culturing under conditions that induce nuclease or ligand synthesis. cDNA is most easily obtained by extracting RNA from cells. can.

Streptavidin [^ rgRrans et al + l1ucleic Ac1ds Re5earch 14 (N4) + 1871-1882 (1986)], Abijito mKul omsa et al., J, Ce1l Biocheg+, 5upp, part 2: 210 (198g)], human hepatitis B antibody, Hon (et al., Korean J. Biochem, +8(1):7-18 (1986)], human aldehyde Dodehydrogenase [Hsu, L, C, et al., Proc, Natl, Aca d, Sci, USA 82: 3771-3775 (1985)], Fib Hong l Cutin [5uzuki, S, et al., Eur, 11o1. Btol, Organ , j, 4-: 2519-2524 (19115) Ko, q Toes Trogen receptor gene [1alLer, P, et al., Proc, NaLl, Ac ad, Sci, USA 82: 7889-7893 (1985)], ]Organization Type plasmi/-gen activator Penn1ca, D, et al., Nature 3 01:214-221, (191!2)] and human term placenta alkaline Sphatase complementation DN A [Kam, 1. and others.

Proc, Natl, Acad, Sci, USA 82: 8715-8719 (1985)] has been successfully cloned.

Although many nuclease genes have been cloned using various methods, this These genes contain extracellular nuclei of 5erraLia-Marcascens. Zeball et al., Gene 57:183-192 (1987)], Bacte et al. 5° exonuclease of lyophage T5 [KaLiman et al., FEBS] Letters 195: 61-64 (1986)] and canine type 2 adhesion. Novirus endonuclease [5pibey et al., J. GenJirol, 70(1): 165-172 (1989)]. l1t son [Gene 74:28 to -289 (198B)] is a restriction endonucleus. Four steps for isolating and cloning lyase and modified methylase genes discloses a method.

Cloning of the nuclease gene and the use of ligands (streptavidin, antibody) All of the above documents describing the cloning of antibodies (Vidins and Antibodies) are incorporated herein by reference. shall consist of:

Another method of cloning nuclease or ligand genes involves DNA or Prepare a library of expression vectors by cloning cDNA. Ru. This library is then tested with nucleases or ligands or A polypeptide having the same amino acid sequence as a fragment or variant of has a nucleotide sequence that can be coded and binds to anti-nuclease or anti-ligand antibodies. Screen for library members that can express matching proteins.

B. Fusion of nuclease and ligand genes Clones obtained by the above method ``Hybrid'' by functionally linking nuclease or ligand genes. Nuclease-ligand fusion genes can be obtained. Entire structure including active site Genes or portions can be fused in any combination and in any order.

The only requirement is to functionally link these structural genes or structural gene fragments. It is to let Next, the obtained no\ib'Jyd gene is transformed into an expression vector. The nuclease of the present invention Produce the ligand fusion protein. Methods for creating fusion proteins are within the skill of the art. It is well known. For example, Murphy (U.S. Pat. No. 4,675,382) teria toxin and polypeptide ligands (hormones, etc.) ＼britado protein discloses a recombinant DNA method for producing quality proteins.

Gene fusions or fragment fusions thereof can be constructed according to conventional methods. can. Such methods include blunt or cohesive ends for ligation, and appropriate termination. Restriction digestion to obtain ends, filling in cohesive ends if necessary, avoiding undesired conjugations. treatment with alkaline phosphatase to ensure stability, and ligation with appropriate gauze. match. Such operations are disclosed by ManiaLis, T. et al. and are well known in the art. It is well known.

Immunoglobulin genes and other genes (staphylococcal nuclease, mouse tumor genetics) child C-1ye, and the Klenow fragment of E. coli DNA polymerase 1. A fusion protein is created by recombinant DNA methods by fusing it with [Neuberger et al., Nature 312: 604-612] (19B4); Neuberger et al., Trends in@Bi.

chemical 5science, 347-394 September 1985] ,margin) Expression of a C nuclease-ligand fusion gene operably linked to the ligand gene nuclease genes or at least parts of these genes; is operably linked into an expression vector, introduced into a host cell, and expressed by the cell. protein can be expressed.

Two DNA sequences (e.g. promoter region sequence and desired fusion protein) coding sequences), the nature of the bond between the two DNA sequences is such that (1) (2) does not result in the introduction of a soft mutation; (3) does not interfere with the ability of the promoter region sequence to transcribe the gene sequence; affects the ability of the desired protein gene sequence to be transcribed by the promoter region sequence. If they do not interfere, they are said to be functionally connected.

The DNA sequence encoding the nuclease-ligand fusion protein is prepared in a conventional manner. Therefore, it can be recombined with vector DNA. The present invention applies to both prokaryotic and eukaryotic This involves expression of the desired fusion protein in the wl cells. For eukaryotic hosts , yeasts (especially Saccharomyces), fungi (especially ^spergil) lus), in vivo or in tissue culture in mammalian cells (e.g. human or primate cells, etc.).

Yeast and mammalian cells also undergo post-translational peptide modifications including glyphsylation It gives you a big advantage in that you can. desired protein in these hosts. A strong promoter sequence and high There are numerous recombinant DNA methods that utilize copy number plasmids.

Yeast recognizes the leader sequence of the cloned mammalian gene product and - secretes a peptide (i.e., a prepeptide) having the sequence. Mammalian cells are post-translational modifications to protein molecules (correct folding or at the correct site) (including glyphsylation).

Mammalian cells useful as hosts include fibroblasts such as VERO or CHO-Kl. Includes cells of cellular origin or derivatives thereof. the desired fusion for the mammalian host. Several possible vector systems are available to express synthetic proteins. . A wide variety of transcriptional and translational control sequences can be used depending on the nature of the host. can. These transcriptional and translational regulatory signals can be expressed by adenovirus, bovine papilloma can be derived from viral sources such as macavirus, monkey virus, etc. (here (the regulatory signals are linked to specific genes with high levels of expression).

Alternatively, derived from mammalian expression products such as actin, flagens, myosin, etc. A promoter can be used. Transcription initiation that can be repressed or activated Regulatory signals can be selected to modulate gene expression. important sig Null is a temperature control whose expression can be suppressed or started by changing the four sentences. under the control of temperature-sensitive regulatory signals or chemical regulation (e.g., intermediate metabolites). It is a regulatory signal that

Expression of the desired fusion protein in a eukaryotic host requires the use of eukaryotic regulatory regions. do. Typically, such regions contain sufficient promoter region to initiate RNA synthesis. It probably contains the area. Preferred eukaryotic promoters include mouse metallo5 (19g2) SV40 early promoter [Benoist, c, et al+ NaLure (London) 290: 301: 5951-5955 ( 1984)].

As is widely known, the translation of eukaryotic mRNA encodes the first methionine. starts at the codon that is For this reason, eukaryotic promoters and the desired fusion The bond between the DNA sequences encoding the protein may feed the methionine. to ensure that it does not contain any intervening codons (i.e. AUG). It is preferable to The presence of such a codon may result in the production of a fusion protein. (If the AtJG fudon is in the same sequence as the desired fusion protein-encoding DNA sequence) ), or as a result of a frameshift mutation. (If the ΔUG codon is in the same reading frame as the desired fusion protein encoding sequence) (when not in frame).

Expression of hormone receptor molecules can also be carried out in prokaryotic cells. preferable Prokaryotic hosts include E, ccli (E. coli), Bacillus, and SLrept. omyces, Rgeudosonas, Salmonella, 5erra Includes bacteria such as Lia. The most preferred prokaryotic host is E. coli. especially Important bacterial hosts include E. coli and 12 strains of HMS174 (F-1rec A, r' x+tm″x, -Rif11) and BL21 (F\ompT, r -5m- 5) and pond intestinal bacteria (Salsonella Lyphimuriu). or 5erratia @arcescen++) and various Pseudom onasfi included. Prokaryotic hosts contain Replico V and Must be compatible with the control sequence.

Prokaryotic cells (e.g., Escherichia coli, B. 5ubtilis, Pseudomonas , StrepLomyceg, etc.) to express the desired receptor molecule. For this purpose, the sequence encoding the desired fusion protein is linked to a functional prokaryotic promoter. It is necessary to be functionally linked to the controller. Such promoters are constitutive or more preferably regulatable (i.e. inducible or disinhibitory) good. Examples of constitutive promoters include bacteriophage-sized l11 (promoter and the bla promoter of the b-lactamase gene of pBR322. is included. Examples of inducible prokaryotic promoters include the main promoter of bacteriophage λ. Right and left promoters (PL and P, ), E. coli trp, recA , 1. acZ, Iacl Sgal and Lac promoter, a-ami [Lllsanen, I et al., J. Bacteriol, 162: 176-182 (1985)], B, 5-28-specific protein of 5ubLilis. Motor [Gilman, M, Z et al., Gene 32: 11-20 ( 1984)], Bacillus bactiliopha Gen, Genet, 20 3:468-478 (1986)]. The prokaryotic promoter is G lick, B.

R, [J, lnd, Microbiol, L: 277-282 (1987 )]; Cenatiempo, Y, [Biochimie@68: 505- 516 (1986)]; and GOtteslian, S., [^nn, R. ev, Genet, 1g+ 415-442 (1984) n outline are doing.

Proper expression in prokaryotic cells also requires liposomes upstream of the gene-encoding sequence. The presence of a protein binding site is required. Such liposome binding sites include, for example, G.

ld, L, et al. [Ann, Rev. Microbiol, 35: 365-40 4 (1981)].

Desired receptor fusion protein hooding sequence and operably linked promoter into either a recipient prokaryotic or eukaryotic cell, either by linear molecules or by covalent bonds. Non-replicative DNA (or can be introduced as an RNA) molecule. Such molecules replicate autonomously expression of the desired receptor molecule is a temporary It will occur by expression. Permanent expression also This may occur by incorporation into the color bodies.

In one embodiment, the desired gene sequence can be integrated into the host cell chromosome. Use vectors that can be used. Cells that have stably integrated the introduced DNA into their chromosomes are one or more markers that allow selection of host cells containing the expression vector. It can also be selected by introducing This marker determines the nutritional requirements of the host. (e.g. 1eu2 or ura3), with normal yeast auxotrophic markers. ), biocide resistance (e.g. antibiotic or heavy metal resistance such as copper), etc. can do. This selection marker gene is a DNA gene to be expressed. It can be directly linked to child sequences and can also be simultaneously transfected with silane. can also be introduced into the same cell.

In a preferred embodiment, the introduced sequence establishes an autonomous replication strategy in the recipient host. Important factors when choosing a viral or viral vector include: Recognize vector-containing recipient cells and select from vector-free recipient cells. The desired copy number of the vector in a particular host; and the ability to “shuttle” vectors between host cells of different species. Whether it is desired or not.

A full range of yeast gene expression systems are available. Such expression vectors Examples of tars include yeast 2 micron circle, expression plasmid YEP13, YCP and YRP, or derivatives thereof. Such a plasmid Well known in the art [BoLsLein, D. et al., Mia+lIi Wnt r, Sy+llp, 19: 265-274 (198Q); Broach, J. R., Molecular Biology of the Yeast Saccharomyces: La. "If Cycle and Genetic Properties", Co1d Spring tlarbo r Laboratory, Co1d Spring l1arbor, N Y, blow A445-4 70 (1981): Broach, J, R, Ce1l 28: 203-2 04 (1982)].

For mammalian hosts, several possible vector systems are available for expression.

One vector type is bovine papillomavirus, polyomavirus, and adenomavirus. viruses or derived from animal viruses such as the 5V4Q virus. Utilizes DNA elements that provide extrachromosomal plasmids. The second vector type is is based on the integration of the gene sequence into the host chromosome. Dye the introduced DNA Stably integrated cells into chromoplasts allow selection of host cells containing the expression vector. can also be selected by introducing one or more markers that .

This marker provides an auxotrophic host with prototropy, biocidal resistance (e.g. antibiotic It is possible to give items such as gold or heavy metals such as copper [). This selection marker gene can be directly linked to the DNA sequence to be expressed, or can be linked directly to the DNA sequence to be expressed. They can also be introduced into the same cell by time transformation. In addition, the optimal Additional elements may be required for composition. These elements include contains transcriptional promoters, enhancers, and termination signals. Ru.

Preferred prokaryotic vectors include, for example, plasmids capable of replicating in E. coli, e.g. Contains pBR322, Co1E1, psclor pACYC184, WVX, etc. be caught. Such a plasmid can be prepared, for example, by −aniat is+T, et al. Molecular cloning, laboratory manual, Co1d Spring l1a rbor Press, Co1d Spring l1arbor, NY (P9 82) Ko has disclosed. Bacillus plasmids include pC194 and pC2. 21, pTl 27, etc. Such plasmids were prepared using Gryczan , T., [Molecular Biology of rBacilli], ^ academic Press , NY (1982), pp. 307-329]. appropriate S The Lreptomyces plasmid contains pIJIO1 [Kendal L, K, J, et al., J, Bacteriol, 169: 4177-4183 (19g?)], and Streptomyces bacteria such as φC31. Phage [Chater, K, F et al., rAcLinomycetales During the 6th International Symposium on Biology J, ^kade+＊1aiKaido, B udapest, llungary (1986), pp, 45-54] included. The P@eudo*onas plasmid was developed by John, J., F et al. [R ev, Inrect, Dis, P: 693-704 (1986)], and Izaki, j, [! pn, J, Bacterio1.33:729-742 (197g)] is approximately I am explaining.

Once the vector or DNA sequence containing the construct has been prepared for expression, the DNA construct The construct can be introduced into a suitable host. Various methods, e.g. using fusion, calcium phosphate precipitation, electroporation, or other conventional methods. can be done. After fusion, cells are grown in culture and screened for appropriate activity. -ning. Expression of the sequence results in the production of a fusion protein of the invention. Ru.

D. Isolation of Nuclease-Ligand Fusion Proteins Fusion Protein Molecules of the Invention extraction, precipitation, chromatography, affinity chromatography, electrophoresis, etc. The recombinant molecules described above can be isolated and purified according to any conventional method. Melt The present invention was developed using affinity chromatography directed toward the ligand component of the protein. Preferably, the fusion protein is purified. [Analy tical Biochemistry 171: 1-32 (198g)] checking ... In addition, a large number of carriers are available commercially - strebtoapidin. Matrinox is known.

According to the present invention, through an assay to detect the presence of a nuclease fusion protein, can be used during routine biochemical purification methods to determine the presence of these enzymes. .

The nuclease fusion proteins of the invention are based on cleavage of their nuclease substrates. It can be identified by As a substrate, e.g. adenovirus-2 (Ad-2) DNA can be used. li in the fusion protein? KL, after this group'1 i (DNA or RNA) using a slow-li type agaronate, which is commonly used to separate digestion products. Separate by electrophoresis in a soft gel. of ultraviolet light in the presence of ethidium bromide Visualize the substrate.

Table 1 (J-) lists potential ligands for use in the present invention: receptors. Or, it is an example of a combination of a receptor and a ligand. According to the invention, the acceptance Any macromolecule or part thereof that can have binding affinity for a specific ligand may include. Are receptors that recognize specific ligands naturally present? or can be prepared. Receptors according to the invention include proteins and non-proteins. Both molecules of protein may be included. Exemplary receptors include biotin, avidi streptavidin, antigen, antibody, lactin, funshugate, enzyme, group quality, cofactor, inhibitor, cation, anion, hydrophobic site, hydrophobic group, hormone Bay effectors, toxins, transport proteins, vitamins, amino acids, sugars, membranes, and Including, but not limited to, liposomes.

Receptors are catalytically active non-proteins that have affinity for their corresponding ligands. Preferably it is a molecule. The receptors used in the present invention are capable of being digested or hydrolyzed. It may be directly or indirectly bound to the nucleic acid. Indirect binding connects the receptor to a second This is done by attaching either non-covalently or covalently to molecules of Now (this second molecule is inside the DNA or RNA molecule to be cleaved or hydrolyzed) (designed to recognize and bind to a specific recognition sequence). According to another method, DNA or RNA with which this receptor is to be reacted with the fusion protein of the present invention. It may also be attached directly to the A molecule (covalently or non-covalently).

B. Combine 1. Direct connection Direct binding of the receptor to the RNA or DNA molecule to be cleaved or hydrolyzed is This may be accomplished by known chemical or enzymatic means. which receptor is bound to the substrate Depending on what is to be done, different methods are used which are well known to those skilled in the art. For example, a book Biotin as a receptor according to the invention can be prepared by numerous chemical and enzymatic methods. It can be attached to a substrate. For an overview of biotin-nucleic acid interactions, see Wilchek et al. [Analytical Bioche@1try 171 : 1-31 (198B)] (this document forms part of this specification). ).

Using nucleotide-containing receptor molecules, many of which are commercially available, The receptors are isolated from double-stranded DNA or RNA using the standard binoctran-rayon method. can be introduced directly into the In this case, the nucleotides in the double-stranded sequence are Can be substituted with a cleotide-containing acceptor molecule. Biotin is a nicktran by using biotinylated nucleotide triphosphates in conjunction with the slation method. introduced into the growing DNA strand [Langer et al., Proc, Nat. l, ^cad, Sci, LIS^786633-6637 (1981)] . Additionally, piotin-containing compounds are suitable for direct introduction of biotin moieties into DNA. [Forster et al. + Nucleic Ac1d] lRe5.13-745-761 (1985); and Re1sfeld Ra + Bioches, Biophys, Res, Comt 14Q゜ : 519-526 (+987).

Alternatively, a novel strand polymerization method for synthesizing single-stranded DNA or RNA Provides a means for introducing nucleotide-containing acceptor molecules in the chain. reaction mixture By changing the receptor-labeled nucleotide in the compound (i.e., 8- receptor, C-receptor, T-receptor or C-receptor), select a novel chain at the receptor can be labeled accordingly. By this method, the newly synthesized strand is contain receptors only at A, GSC, or T nucleotide positions in Can be done. The use of two or more receptor-nucleotide combinations allows Introducing receptor-containing nucleotides at multiple nucleotide sites in a sequence becomes possible.

This study was carried out by setting up four reactions using different nucleotide-containing receptors. The invention provides methods for sequencing DNA or RNA molecules. According to the invention D To sequence NA or RNA, ASG, C, or Ty, cleotide Each of the four reaction tubes in which the receptor was introduced into the ligand site was Separately incubate with reactions such as time, temperature, and fusion protein concentration. By varying the reaction conditions, a random population of digested molecules is generated in each reaction. - can be obtained in test tubes. The cleavage products of all four reactions are then Analyze by gel electrophoresis according to standard sequencing methods.・Visualize products This new NA or RNA strand can be labeled before or after nuclease digestion Ring the bell. Labels used in accordance with the invention include enzyme labels, radioisotope labels, labels, non-radioactive isotope labels, fluorescent labels, and chemiluminescent labels. , but not limited to these.

2 Indirect connection In addition to direct binding of the receptor to the nucleic acid substrate, the binding of the receptor to the DNA or RNA sequence to be cleaved Receptor molecules can be indirectly bound. This method is most preferred. This aspect In this method, receptors are attached to macromolecules (covalently or non-covalently). ). Well-known chemical and Enzymatic methods can be used. This macromolecule-receptor complex is then The receptor mediates binding of the receptor to the substrate by recognizing and binding to the substrate. Ru. In this way, the receptor is indirectly bound to the nucleic acid substrate. Therefore, according to the present invention The macromolecule receptor is a substrate-macromolecule-receptor that will bind to the fusion protein of the invention. Sometimes a body complex is formed.

The macromolecules used for indirect binding of receptors to substrates are those that are parent to DNA or RNA. (can be an engineered protein or non-protein molecule; macromolecules) Preferably, the progeny have an affinity for specific sequences within the substrate, but not for non-specific DNA. Alternatively, substrates can be randomly digested using RNAΔ binders. Bio Preparation of receptor-macromolecules such as tin-conjugated or enzyme-labeled antibodies is , 11arlow et al. [[Antibodies, Laboratory Manual], Coldspring ) Larbor, IIY (1988)].

Most preferably, the site-specific macromolecule is grouped by hybridization. RNA or DNA nucleotide probes that bind to proteins. That is, the present invention Accordingly, receptors containing single-stranded DNA or RNA macromolecules are known to those skilled in the art. Hybridize to a single-stranded substrate using well-known conventional hybridization techniques. let The preparation of synthetic oligonucleotides with biotin residues attached was described by Chu et al. [D! A4: 327-331 (1985)]. Nucleochi Biotinylation of code sequences and their use in hybridase 7-1 is discussed. Many studies that discuss CAanalytical Bioche +aistry 171:1-32 (19118)] .

■・Sato 4 The sequence-specific cleavage or hydrolysis substances of the present invention can be used for gene cloning, nucleic acid It has applications in structural studies and chromosome mapping. Endonu of the present invention Crease fusion protein can be used to convert any nucleotide sequence RNA or DNA A predetermined site within A can be selectively cut. This thing is available Type 11 restriction endonuclease is engineered to cut any predetermined sequence. This is especially important when considering that it cannot be prepared. Known Limitations Donucleases recognize specific recognition sequences and cleave within or near them. It is only possible to do so. Therefore, these limitations may end up frustrating research efforts. Any predefined number of nucleotides can be removed from the end. known Controlled digestion with xonucleases is possible, but this Only in the general way to obtain molecular populations (i.e., enzyme concentration, time, Controlling hydrolysis by varying temperature and/or enzyme conditions.

Thus, prior to the present invention, nuclei removed during hydrolysis of DNA or RNA It was not possible to specifically control the number of punches. However, according to the present invention, 1 Controlled digestion of single-stranded or double-stranded nucleic acid molecules is performed in a defined manner to Specific ends of the molecule are shortened to a predetermined length.

The defined removal of a certain number of nucleotides from the terminus indicates the presence of undesired sequences. (i.e., lethal or non-functional promoter sequences).

Controlled hydrolysis of the 5′ end facilitates removal of sequences upstream of the ATG initiation fudon. and thus allow for the replacement of the natural regulatory elements of a particular gene.

The invention also provides methods for sequencing DNA or RNA molecules. present invention The usefulness of this feature is self-evident.

Furthermore, in addition to the utility of hybrid enzymes for genetic research, the present invention Provide materials for diagnosing these diseases. For example, caused by a virus infection. This disease results in the introduction of genetic material into the host. According to fusion proteins that recognize these unique heterologous sequences and cleave them can be processed and therefore digestion will not occur in an uninfected host. . Therefore, after reaction with this site-specific fusion protein, DNA samples from the host Viral infection can be determined by analyzing gel electrophoresis of samples. . Many viruses (papillomavirus, hepatitis A virus, hepatitis B virus) , herpes simplex virus, human immunodeficiency virus, HTLV-III, HTLV-1 ゜ Corn Mosaic Virus, Tobacco Mosaic Virus, and Bacteria (including but not limited to phages) into prokaryotic as well as eukaryotic hosts. It can be detected at

Having thus far described the present invention in general terms, it will be appreciated that the present invention will now be described with reference to the following examples. The invention will be more easily understood. These examples are given for illustrative purposes only. Unless otherwise specified, no limitations on the present invention are intended.

Example 1 Chimeric protein of staphylococcal nuclease and streptavidin creating quality Streptavidin- or avidin-pyotin complex is abnormally high affinity (k, l=IQ-15M) and hence the number of non-covalent interactions between two compounds. Extremely versatile in a wide variety of bioanalytical applications, primarily due to its high stability It is very useful as a general mediator of [Au5ubel. P, l! , et al., “Recent Protocols in Molecular Biology J + Greene Publish ing As5ociates and 1iley-1nLerscienc e+ mew York (19B?)]. Many piotin-containing derivatives have a nucleated piotin moiety. Some of them have been found suitable for direct introduction into the octide sequence. is available commercially. In addition, the conventional nick translation method or is biotin, which can be introduced into DNA or RNA molecules by the chain reaction method. Polymerized oligonucleotides are available. DNA or RNA (i.e. When a plasmid, chromosome, etc.) is labeled with a pyotin receptor, the receptor sequence is This site acts as a specific target, which is then recognized and further stimulated Can interact with avidin or avidin-containing fusion proteins .

Figure 1 shows a chimeric protein of staphylococcal nuclease and streptavidin. This shows the construction of a hybrid gene coded by pFOG301 is a block Contains the NUC gene that harbors Daphylococcal nuclease [5hor tle.

D., Button η 181-189 (1983)], pTSA-1 is a streptoa Contains the gene that feeds Vidin [5ano et al., Proc, Na tl, Acad, Sci, 87: 142-146 (1990) and Ar garana et al., Nucleic Ac1ds Re5earch 14: 1871-1882 (1986)].

Amplification of the NUCJl gene and Ndel regulation to the 5° end of the amplified NUC gene The introduction of the restriction site and the introduction of the Xba1 site at the 3° end was performed using pFOG301 plus. The following oligonucleotides were used as primers to perform PCR to amplify the DNA. Do: 5' -TTAG^Edited TCCATATGACAG^^TACTTATTAA GT-3' (SEQ ID NO:l) and 5°-TTAC^^TGATCTAGATTGACCTG^^TCAGCGTT -3° (SEQ ID NO: 2) is used. PCR was performed using the manufacturer's method (Perkin - Elmer). 95°C (1 minute), 45°C (1 minute), 75°C ( 2 minutes) for approximately 30 cycles. 5 μm out of 100 μm of PCR reaction mixture Analyze to determine the success of the PCR reaction. The remaining reaction mixture was run on a 1% agarose gel. The PCR product was purified according to the manufacturer's method (ieneclean [BIo1 01 Inc, La Jolla, C^].

Amplification of the gene that feeds streptavidin and 5 of the amplified genes The introduction of the Xba1 site at the ' end and the introduction of the Ba81 site at the 3' end is A protocol for performing PCR to amplify the streptavidin gene derived from TSA-1. As a primer, the following oligonucleotide 5°-GCCCTCTAGAGAG Performed using GCCGGCATCACCGGCACC-3' (SEQ ID NO: 3) . PCR is performed according to the manufacturer's method (Perkin-El SR). PCR The product is purified with Geneclean as described above.

Next, the amplified NUC gene was incubated with Xbal and Ndel at 37°C for 2 hours. , the amplified streptavidin gene was extracted with Xbal and BagiHl. ℃ for 2 hours, and the pET3a plasmid was incubated with Ndel and Ba5HI for 3 hours. NUCilli and Streptavia by digestion separately for 2 h at 7 °C. The PCR product of the gene gene was cloned into the pET3a expression vector (Novagen). Make it into a loan. All of these reactions were performed with phenol/C11C1 extraction followed by Stop by ELOH precipitation and 70% EtOH wash. then digested Lyophilize NtJC and streptavidin DNA. pET3 converted to ch The aDNA was lyophilized and then single molecule cloning using the standard method Draniatls et al. , Laboratory Manual J, 2nd Edition, Co1d Spring 1larbor (19119) Dephosphorylation and cleaning according to E. Then, the digested N The tJC gene and streptavidin gene were extracted using the conventional method [Maniatis; Connect to the digested and dephosphorylated pET3a expression vector using the above method. . Using a portion of the ligation reaction mixture, the HMSt74 cell line [F-1 hsdR, reeA, Rtf”; Ca5pbe11 et al., Proc, NaL l, Acad, Sci, USA 75: 2276-2280 (1978)] Transform. Take several clones and grow them in LB medium containing ampicillin. Grow in the evening. Plasmid DNA was obtained from each culture and Ndel/Baa Analyze by HI restriction digestion. Choose a plasmid that has the correct insert size , this DNA is used for expression of the fusion gene. Transfer this plasmid DNA to pETNA Name it S-1oo (Figure 1).

Example 2 Chimera containing staphylococcal nuclease and streptavidin Protein expression and purification The structural gene of the chimeric protein (Figure 1) is expressed in E. coli. Common law [Man iatiS, supra] using pENS-100 to generate BL21(DE3)[F -10@pT, hsdS.

gal; 5tudier et al., J. Mo1. Biol, 189: 113- 130 (1986) and Grodgerg et al., J. riol, 170: 1245-1253 (1988)]. B L21 is available from Novagen. pETNS-100 plasmid BL21(DE3) to be retained was added to 0.4% glucose and 50 μl/ml aliquots. Grow at 37°C with shaking in LB medium supplemented with ampicillin. Ac0 When 0 reaches 1.0, add 100mM IPTG to a final concentration of 0.4+nM. In addition, the T7 RNA polymerase gene under the control of the IacU V5 promoter induce. After induction, cells were incubated for an additional 2 hours at 37°C without shaking. ESano et al., l’roc, Natl, Acad, Sci, 87 : 142-146 (199111)].

All operations for purifying the fusion protein are performed at 4°C. The above guidance Harvest the culture (100 ml) by centrifuging at 1600 x g for 10 min. Ru.

The cell pellet was dissolved in 100+nM NaCl, bsM EDTA, 10mM). Wash with HCI (pH 8,0) (XOO+mD) and centrifuge as above. cells were treated with egg white lysozyme (IIIg) and 0.1% τriLon (2+M EDTA containing 10, 30mM Tris-HCI (pH 8,0) (10 B? ). Mg5O, (1,0M), DNase l (1sg/ml) and RNase A (1 mg/at) at 12 mM, 10 ug/’ml, and and to a final concentration of 10 μg/liter and leave the mixture at room temperature for 15 minutes. . This cell lysate was prepared in 39. Centrifuge for 15 minutes at room temperature in OOOxg to remove the precipitate. Part 2 Tsuru MEDTA, 30mM Tris-HCI (pH 8.0), 0.1% Tri Wash with tons of X-100 (l Oml) and then centrifuge as above. child 6M guanidinohydrochloric acid (pH 1, 5X5+el) with gentle stirring of the precipitate. Guanidine was dissolved in Remove HCI. This dialysate was transferred to 39,000 x g for 15 embryos kl-, A-c-i. -, β7. +142-146 (+990)].

This chimeric protein was further purified by biotin-affinity column as described above. Purify [Wilchek, 11 et al., Ansl, Bioehc41'υ: 1-32 (1988)].

Site-specific cleavage reaction to inserted DNA 1 Borisk 17 as shown in Figure 2 Otide substrates (a) and (b) were prepared from circular single-stranded Ml:3+p7 DNA. make 1 mMI3mp7 DNA is Iietgin method [total insertion ads] or Enzymology 101: 20 (1983)], RF Transform colonic TGI cells with ML3sp7 DNA (Yamersham) Prepared by: To prepare group '1(a) (Figure 2), the resulting ring closure 1 vAl) Linearize NA with BamHl [Been & Champous , 1 iethods of Enzy+*ology lot: 90 (1 983)]. Buffer [0,2M NaCl, 1OmM) Lis-)-ICI (pH 7,4), circular DNA (approximately 30 μg) in LsM EDTA] (80 μl), Heated at 95°C for 2 minutes, then at 65°C for 5 minutes. After cooling to room temperature, 0.2M MgC1* (3 μl) and Ba(1 (6 μl; 72 units) ) and then partially incubated at 37°C. This DNA was mixed with 0.8% a Purified on galose gel and dephosphorylated with bovine intestinal phosphatase (BRL) [Maniatis et al., +982]. Then linearize with BIIIHI The 5' end of the DNA was labeled with stP, and the 3' end was labeled with a modified method [Challber g & England, Methods or Enzymology 6 5: 39 (198O) : Been & Champous (1983)]. Brief explanation Specifically, 20+*M) Lis-HCI (pH 7,4), 7% MgCl, 5 0+aM NaCl, 10mM DTT, 0.05mM dGTP, 0.25μM (,α-”P]dATP(＾++erghow, 800Ci/+5ol), and a buffer containing the Klenow fragment of DNA polymerase 1 (10 units) B amHl digested DNA (approximately 4 μg) in (50 μl) was heated at 1I″C for 30 minutes. Incubate for a while.

This DNA is precipitated with ethanol twice to remove unintroduced dATP.

The second group 'Jlf(b) (Figure 2) is located between nucleotides 460 and 460 of the circular single-stranded DNA. 489, the following oligonucleotide 5°-CCCCTC^^^TGCTTTTA^^CAGTTCAGA^^^-3' (SEQ ID NO: 5). Then, the double-stranded Cut the region at Dral. 4uM 3O-nu oligonucleotide, lOaM MgCl, 10mM Tris-HCI (pH 8,0), and 10-M Mg Circular DNA (approximately 20 μl) in buffer containing CIt (45 μl) was heated to 70 °C. and then slowly cooled to 37°C, then dral (51 rivers; 200 units) Add. This reaction solution was partially incubated at 37°C. This DNA is Purify on 0.8% agarose gel and dephosphorylate with calf intestinal phosphatase as lylated and labeled at its 5′ end.A binding site oligonucleotide as shown in Figure 2. Biotide: 5'-CCCGCACJAGCCGCT4° (SEQ ID NO: 6) The conversion is prepared according to the manufacturer's method (C1an, Tech). This biochi Streptavidin prepared in Example 2 using the converted oligonucleotide - the DNA substrate (a or b) to which the nuclease fusion protein is to be cleaved; be indirectly coupled to.

50mM Tris-HCI (p[47,0), 50mM NaCl and 1mM ED Approximately 12*M “P-labeled group @DNA, 14 nM biotinylated oligo in TA Contains nucleotides and 14 nM nuclease-streptavidin chimera The fusion protein was prepared by preparing a reaction mixture (total volume of 10 μl). to cleave the substrate (a or b). This mixture was heated to 65°C for 2 minutes and then Adjust the temperature to the desired temperature. This reaction was ) and after the specified reaction time 10-M ethylene glycol. Contains bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTΔ) The reaction was stopped by adding a formamide-dye mixture (10 μl).

The stopped reaction mixture is heated to 90° C. for 3 minutes and then cooled on ice. profit The resulting DNA fragment was incubated with 5% polyacrylate containing 7M area (l·20 crosslinks). Electrophoresis was performed on a lylamide gel or 1% agarose gel, followed by odak Carry out autoradiography at -80°C using AR5 X-ray film. Analyze by.

The practice of the invention is obvious to those skilled in recombinant DNA technology, enzymology and/or related fields. Modifications of the above embodiments for implementation are intended to be included within the scope of the following claims.

All publications and patent applications mentioned in this specification are incorporated herein by reference. It indicates the technical level of a person skilled in a field. All publications and patent applications are , which constitutes a part of this specification in its entirety.

The present invention has been described in some detail by way of illustration and examples for purposes of clear understanding. However, certain changes and modifications may be practiced within the scope of the claims appended hereto. It should be obvious.

(Hereafter, margin) V) Contact information: (A) Application number: Us (B) Application date/current date (C) Classification: Unspecified (vi) Patent attorney/agent information: (2) Information on array number 1 (i) Features of array (A) Length: 33 base pairs (B) Type: Nucleic acid (C) Number of strands: single strand (D) Topology: linear (ii) Sequence type: DNA (xl) Sequence: Sequence number l; TTAGAATTCCAT＾TGACAGA ATACTTATTA AGT 33(2) Information on array number 2 (i) Array characteristics: (A) Length, 33 base pairs (B) type nucleic acid (C) Number of strands - single strand (D) Topology linear (11) Sequence type: DNA (xi) Sequence Sequence number 2・ TTACAATGAT CT^GATTGACCTG^^TCAGCGTT 3 3(2) Information on array number 3 (i) Characteristics of array (A) Length: 31 base pairs (B) Type: Nucleic acid (C) Number of chains, - heavy chain (D) Topology, linear (ii) Sequence type: DNA (xi) Sequence: Sequence number 3: GCCCTCTAGA GAGGCCGGCA TCACCGGCACC31 ( 2) Information on array number 4 (i) Array characteristics: (A) Length: 32 base pairs (B) Type: Nucleic acid (C) Number of strands: single strand (D) Topology/linear (ii) Sequence type: DNA (xi) Sequence, Sequence number 4: CGCGAGGATCCCTGCTGAACGGCGTCGAGCGG 32 ( 2) Information on array number 5 (i) Array characteristics: (A) Length = 30 base pairs (B) Type: Nucleic acid (C) Number of chains, - heavy chain (D) Topology: linear (ii) Sequence type: DNA (xi) Sequence: Sequence number 5 CCCCTCAAAT GCTTTAAACA GTTCAGAAAA 30 ( 2) Information on array number 6 (i) Characteristics of array:゛ (A) Length: 15 base pairs (B) type nucleic acid (C) Number of chains, - heavy chain (D) Topology/linear (ii) Sequence type: DNA (xi) Sequence/Sequence number 6: CCCGCACAAG CCGCT 15 (2) Information on sequence number 7 (i) Sequence characteristics; (A) Length: 22 base pairs (B) type nucleic acid (C) Number of strands: single strand (D) Topology/linear (ii) Sequence type: DNA (xi) Sequence Sequence number 7: ^TCGTCGTCT GGTA^^CGAG GG 22 NUC gene strike Leptavidin gene front page continuation (51) Int, C1,” Identification symbol Internal office reference number Cl2Q 1/68 Z 7823-4B//(C12N 9/16 C12R1:19) I

Claims (30)

[Claims] 1. Is it a fusion protein that is a hybrid between a ligand and an endonuclease? A site-specific endonucleosome that can selectively cleave any nucleotide sequence, consisting of Rease. 2. The site-specific method according to claim 1, wherein the nucleotide sequence to be cleaved is RNA. Different endonucleases. 3. The site-specific method according to claim 1, wherein the nucleotide sequence to be cleaved is DNA. Different endonucleases. 4. The nuclease is avidin nuclease or a catalytically active fragment thereof. The site-specific endonuclease according to claim 1, which is 5. The ligand is streptavidin or a catalytically active fragment thereof. The site-specific endonuclease according to claim 1. 6. The nuclease is a staphylococcal nuclease or a catalytically active fragment thereof. The site-specific endonuclease according to claim 1, which is an agent. 7. Fusion protein is a staphylococcal nuclease streptavidin conjugate 2. The site-specific endonuclease according to claim 1, wherein the site-specific endonuclease is 8. A fusion protein that is a hybrid between a ligand and an exonuclease? A site-specific exonucleotide that can selectively hydrolyze any nucleotide sequence, consisting of Nuclease. 9. The site-specific method according to claim 4, wherein the nucleotide sequence to be cleaved is RNA. Different exonucleases. 10. The site according to claim 4, wherein the nucleotide sequence to be cleaved is DNA. Specific exonuclease. 11. Method for selectively cleaving arbitrary nucleotide sequences at predetermined sites And, (a) binding a receptor to the nucleotide sequence to be cleaved; (b) binding the receptor to the nucleotide sequence to be cleaved; containing nucleotide sequences with a hybrid between the ligand and the endonuclease. (c) contacting the components of step (b) with a fusion protein containing said receptor; The nucleotide sequence is incubated for a sufficient period of time to cleave within or near the recognition sequence. a method consisting of incubating; 12. The fusion protein is a staphylococcal nuclease streptavidin conjugate. 12. The method of claim 11, wherein the gate is a gate. 13. 13. The method according to claim 12, wherein the receptor is biotin. 14. Method for selectively cleaving arbitrary nucleotide sequences at predetermined sites And, (a) binding a receptor to a macromolecule capable of binding to said nucleotide sequence; (b) binding said nucleotide sequence; (c) contacting a receptor-containing macromolecule with the nucleotide sequence; A fusion protein that is a hybrid between a ligand and an endonuclease contact with: and (d) cutting the component of step (c) within or near the recognition sequence of the sequence; incubate for sufficient time; A method consisting of things. 15. The macromolecule uses oligonucleotides that are complementary to the part of the nucleotide sequence that it intends to cleave. 15. The method according to claim 14, wherein the nucleotide is a nucleotide. 16. 16. The method according to claim 15, wherein the receptor is biotin. 17. The fusion protein is a staphylococcal nuclease streptavidin conjugate. 17. The method of claim 16, wherein the gate is a gate. 18. Method for selectively cleaving arbitrary nucleotide sequences at predetermined sites And, (a) binding a receptor to the nucleotide sequence to be cleaved; (b) binding the receptor to the nucleotide sequence to be cleaved; contacting the containing nucleotide sequence with an anti-receptor capable of binding to the receptor; (c) combining the receptor-antireceptor-containing nucleotide sequence with a ligand and an endonuclease. and (d) step ( component c) within the recognition sequence of the receptor-antireceptor-containing nucleotide sequence or a method consisting of: incubating for a sufficient period of time to allow cleavage in its vicinity; 19. the removal of a specified number of nucleotides from the end of any nucleotide sequence A method for hydrolyzing the sequence by (8) attempting to hydrolyze the sequence. (b) binding a receptor to the nucleotide sequence; (b) binding the receptor-containing nucleotide sequence; , a fusion protein that is a hybrid between a ligand and an exonuclease. and (c) adding the components of step (b) to the receptor-containing nucleotide sequence. Incubate for sufficient time to allow water to split; A method consisting of things. 20. the removal of a specified number of nucleotides from the end of any nucleotide sequence 1. A method for hydrolyzing said nucleotide sequence by (a) (b) binding the receptor to a macromolecule capable of binding; (b) binding the receptor-containing macromolecule to the receptor; (c) contacting the components of step (b) with the ligand and exonuclease; contacting with a fusion protein that is a hybrid between lyase; and (d) incubating the components of step (c) for a sufficient time to hydrolyze the sequence; do; a method consisting of doing; 21. the removal of a specified number of nucleotides from the end of any nucleotide sequence A method for hydrolyzing said sequence by: (a) attempting to hydrolyze it; (b) binding a receptor to the nucleotide sequence; (b) binding the receptor-containing nucleotide sequence; , contacted with an anti-receptor capable of binding to the receptor; (c) combining the receptor-antireceptor-containing nucleotide sequence with a ligand and an exonuclease. and (d) step ( component c) sufficient to hydrolyze the receptor-antireceptor-containing nucleotide sequence; incubate for a period of time; A method consisting of things. 22. A method for producing a DNA or RNA fragment, comprising: (a) cleaving (b) binding the receptor to the DNA or RNA molecule of interest; (b) binding the receptor-containing D; NA or RNA is a hybrid between a ligand and an endonuclease (c) bringing the components of step (b) into contact with the receptor-containing DNA or or RNA for a sufficient period of time to cleave; and (d) obtaining a cleaved DNA or RNA fragment from step (c); A method consisting of 23. A method for producing a DNA or RNA fragment, the method comprising: (a) (b) binding the receptor to a macromolecule capable of binding to the leotide sequence; contacting the large molecule with the DNA or RNA molecule to be cut; (c) The components of step (b) are combined into a hybrid between the ligand and the endonuclease. contacting with a fusion protein that is; (d) applying the components of step (c) for a period of time sufficient to cleave the DNA or RNA molecule; incubate; and (e) obtaining a cleaved DNA or RNA fragment from step (d); A method consisting of 24. The macromolecule uses oligonucleotides that are complementary to the part of the nucleotide sequence that it intends to cleave. 24. The method according to claim 23, wherein the nucleotide is a nucleotide. 25. 25. The method according to claim 24, wherein the receptor is biotin. 26. The fusion protein is a staphylococcal nuclease streptavidin conjugate. 26. The method of claim 25, wherein the gate is a gate. 27. A method for producing a DNA or RNA fragment, comprising: (a) cleaving (b) binding the receptor to the DNA or RNA molecule of interest; (b) binding the receptor-containing D; contacting the NA or RNA with an anti-receptor capable of binding to the receptor; (c) The receptor-antireceptor-containing DNA or RNA is combined with a ligand and an endonuclease. (d) step (c) for a period sufficient to cleave the receptor-antireceptor-containing DNA or RNA. and (e) incubating the cleaved DNA or RNA fragments. obtained from step (d); 28. 1. A method for sequencing DNA or RNA, comprising: (a) DNA or RNA sequencing; receptors for specific types of nucleotides A, G, C or T throughout the NA sequence. combine; (b) placing the receptor-containing nucleotide sequence between the ligand and the endonuclease; (c) contacting the components of step (b) with a fusion protein that is a hybrid; incubating for a sufficient period of time to obtain a random population of aborted products; and (d) separation of the nucleotides by separating the cleavage products by gel electrophoresis; A method consisting of comparing the cleavage products of each type. 29. 29. The method of claim 28, wherein the receptor is biotin. 30. The fusion protein is a staphylococcal nuclease streptavidin conjugate. 30. The method of claim 29, wherein the gate is a gate.