AU1972392A - Universal site-specific nucleases - Google Patents

Description

UNIVERSAL SITE-SPECIFIC NϋCLEASES

Field of the Invention

The invention relates to the field of fusion proteins and their use as site-specific nucleases.

Specifically, the fusion proteins of the present invention are ligand-protein hybrids composed of a ligand-exonuclease or a ligand-endonuclease.

Background of the Invention Restriction endonucleases are a class of enzymes that occur naturally in prokaryotic and eukaryotic organisms. When they are purified away from other contaminating cellular components, restriction endonuclease can be used in the laboratory to cleave double stranded DNA molecules into precise fragments. This property enables DNA to be uniquely identified and to be fractionated into constituent genes.

Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the "recognition sequence") along DNA. Once found, they cleave DNA within, or to one side of, this sequence. Different restriction endonucleases have affinity for different recognition sequences. About 100 kinds of different endonucleases have so far been isolated from many microorganisms, each being identified by the specific base sequence it recognizes and by the cleavage pattern it exhibits.

Although restriction enzymes are frequently used by the genetic engineers to manipulate DNA in order to create novel recombinant molecules, the site-specificity of these enzymes tends to limit the usefulness of these genetic tools. Often, the unavailability of endonucleases which cleave any desired recognition sequence frustrates research efforts. Moreover, studies of RNA structure and function have also been hampered by the failure to isolate an analogous class of RNA restriction enzymes from nature.

To address this problem, oligonucleotides carrying reactive groups have been developed during recent years to direct speci ic reactions at pre¬ selected sequences on both single stranded and double stranded nucleic acids. Several metal chelates such as: Fe-EDTA (Boutorin, A.S. et al.. FEBS Lett. 172; 3-46 (1984); Chu et al.. Proc. Natl. Acad. Sci. USA 82: 963-967 .(1985) ; Dreyer, G.B. et al.. Pros. Natl. Acad. Sci. USA 82:968-972 (1985); Boidot-Forget, M. et al. _f Comptes Rendue Acad. Sci. Ser. 2.(302) :75-80 (1986); Boidot-Forget, M. et al. , Gene 72:361-371 (1988)); Cu-phenathroline (Chen et al.. Proc. Natl. Acad. Sci. USA 83_:7141-7151 (1986); Chen et al.. J. Am. Chem. Soc. 110:6570-6572 (1988); Francois, J.C. et al.. Biochemistry 27:5891-5896 (1988); Francois, J.C. et al.. Proc. Natl. Acad. Sci. USA 86:9702-9706 (1989) ; Francois et al.. Proc. Natl. Acad. Sci. USA 86:9702-9706 (1989)); and Fe-porphyrins (Le Doan et al.. Nucleic Acids Res. 15:8643-8659 (1987); Le Doan et al.. Nucleic Acids Res. 25:8643-8659 (1987)); as well as Staphylococcal nuclease and RNase-S induce cleavage reactions at specific sequences when they are tethered to oligonucleotides (Le Doan, T. et al. , Nucleic Acids Res. 15:7749-7760 (1987) ; Praseuth, D. et al.. Proc. Natl. Acad. Sci. USA 85.:1349-1353 (1988); Lee et al.. Biochemistry 27:3197-3203 (1988); Praseuth et al.. Biochemistry 27:3031-3038 (1988)).

Corey et al. rBiochemistrv 28_:8277-8286 (1989)) has successfully developed a biological catalyst with novel speci icity by introducing a new binding domain to the enzyme. A "hybrid nuclease" was generated by fusing an oligonucleotide to a unique site on the relatively non-specific phosphodiesterase,

SUBSTITUTE SHEET staphylococcal nuclease. Such a hybrid nuclease was able to selectively cleave single-stranded M13mp7 DNA under a much wider range of conditions than was previously possible (Le Doan et al.. Nucleic Acids Res. 15:7749-7760 (1987); Praseuth, D. et al. , Proc.

Natl. Acad. Sci. USA 85:1349-1353 (1988); Lee, B.L. et al.. Biochemistry 27:3197-3203 (1988); Praseuth, D. et al.. Biochemistry 27:3031-3038 (1988)).

fimrπnaT-y of the invention

The present invention is directed to a site- specific endonuclease capable of selectively cleaving any nucleotide sequence. The site-specific endonuclease of the present invention is a fusion protein composed of a hybrid between a ligand and a nuclease.

Preselected nucleotide sequences may be selectively cleaved according to the present invention by first binding (covalently or non-covalently) a receptor to a particular site within the nucleotide sequence to be cleaved, such receptor being specific for the ligand of the ligand-endonuclease fusion protein. The ligand constituent of the ligand- endonuclease fusion protein of this invention is attached to the receptor within the nucleotide sequence due to the affinity shared between the ligand and the receptor. In this manner, by contacting the receptor containing nucleotide sequence with the fusion protein of the present invention, one or a number of preselected sequences can be digested, thereby fragmenting the original nucleotide sequence at the desired sites.

The present invention is specifically directed to a fusion between a Staphylococcal nuclease and a streptavidin. Such a fusion protein recognizes biotin as a receptor through a streptavidin-biotin interaction. The biotin may be attached directly or indirectly to the nucleotide sequence to be cleaved by the fusion protein. Preferably, the biotin is bound to a macromolecule such as a oligonuclotide which is complimentary to a portion of the nucleotide sequence. By binding (hybridizing) the biotin-oligonucleotide to the nucleotide sequence, the Staphylococcal nuclease-streptavidin fusion protein of the present invention can cleave the nucleotide sequence in a si±e directed manner.

The present invention is further directed to site-specific exonucleases. In this embodiment, fusion proteins consist of a hybrid between a ligand and a exonuclease. Use of this site-specific fusion protein in accordance with the present invention allows for controlled exonuclease digestion of the nucleotide termini. Accordingly, defined sequences may be removed (hydrolyzed) in a controlled manner, producing a shortened nucleotide fragment.

In another embodiment of the invention, the receptor containing nucleotide sequence is first bound to its corresponding antireceptor, forming a receptor- antireceptor complex attached to the nucleotide sequence. The antireceptor component of this complex is recognized by the ligand portion of the fusion protein, thus attaching the hybrid nuclease to the DNA or RNA substrate to be cleaved. The present invention also is directed to the digestion of DNA or RNA substrates by indirectly binding the receptor to those molecules. Thus, rather than binding the receptor directly to the sequence to be cleaved, the receptor is attached to a macro- molecule which has affinity for the substrate. In this embodiment, the macromolecule "bridge" between the substrate and receptor may be designed to randomly attach to the substrate or bind to specific recognition sequences within the DNA or RNA substrate.

Brief description of the drawings

Figure 1 shows the construction of a vector of expressing the nuclease-streptavidin fusion protein.

Figure 2 shows the preparation of DNA substrates which are cleaved by the nuclease-streptavidin fusion protein.

Description of the Preferred Embodiments

Definition

In the description that follows, a number of terms used in recombinant DNA technology are extensively utilized. In order to provide a clearer and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

Fusion protein. As used herein, the term "fusion protein" is meant to refer to two or more macromolecules, one of which is a nuclease, that are functionally linked to form a single macromolecule. By "functional linked" is meant the linkage (covalently or non-covalently) of each macromolecule in such a manner that the catalytic or structural activity of each macromolecule is retained in the fusion protein. For example, the fusion protein made up of a hybrid between a nuclease and a ligand will retain the nuclease activity and the binding activity or at least some portion of those activities in the fusion protein. In addition to a nuclease as one of the components of a fusion protein, at least one ligand is included. Furthermore, one or more ligands and nucleases may make up the fusion protein of the present invention.

Ligand. As used herein, by "ligand" is meant any macromolecule or portion thereof which possesses an inherent a finity for a given receptor (antiligand) . The only limitation in accordance with present invention is that the term "ligand" can not encompass a nucleic acid molecule, including RNA and DNA. Affinity. As used herein, by "affinity" is meant to refer to the natural attraction, either biochemical, chemical or physical in nature, between the ligand, and receptor or antireceptor. This attraction results in covalent or non-covalent binding of a ligand to its corresponding receptor or corresponding antireceptor. Furthermore, a receptor has affinity for its corresponding antireceptor.

Receptor (antiligand^ ♦ As used herein, the term "receptor" is meant to include any macromolecule or portion thereof capable of recognizing (having a binding affinity to) a particular ligand. Receptors which recognize a particular ligand naturally exists or can be prepared. Illustrative receptors include, but are not limited, biotin, avidin, streptavidin, antigens, antibodies, lactins, glycoconjugates, enzymes, substrates, cofactors, inhibitors, cations, anions, hydrophobic sites, hydrophobic groups, hormones, effectors, toxins, transport proteins, vitamins, amino acids, sugars, membranes, liposomes, etc. By catalytically or biologically active receptor is meant to refer to a receptor which is capable of recognizing and binding to its corresponding ligand.

Anti-receptor. In some situations, a anti¬ receptor can serve a dual function of binding to a receptor which is attached to the nucleotide sequence and then serving as a receptor to the ligand portion of the fusion protein, such that the ligand and the receptor, which can not bind directly to each other, are joined by the antireceptor, providing a linkage between the ligand and the receptor.- Substrate. Substrate as used herein refers to the DNA or RNA molecule,to be cleaved or hydrolyzed by the fusion protein of the present invention.

Fragment. A "fragment" is meant to refer to a portion of the complete macromolecule (ligand, receptor, antireceptor, or nuclease). By - a "catalytically or biologically active fragment" of these macromolecules is meant a fragment which retains all or some of the catalytic or biological activity possessed by the complete molecule, especially activity that allows specific ligand/receptor binding or sequence cleavage by the molecules of the invention.

Variant. A "variant" of a macromolecule is meant to refer to a ligand, receptor, antireceptor, or nuclease substantially similar in structure and catalytic activity to its native molecule or fragment thereof, but not identical to such molecule or fragment thereof. A "variant" is meant to include isozymes and alleles. A variant is not necessarily derived from the native molecule. Thus, provided that two molecules possess a similar structure and catalytic activity, they are considered variant as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of these molecules is not identical to that found in the other.

I. Fusion Protein Catalytic Activities

The present invention provides for a site-specific endonuclease which is capable of cleaving within the sequence of a nucleic acid molecule (RNA or DNA, single- or double-stranded) at any or a number of preselected sites. Furthermore, the present invention is directed to a site-specific exonuclease which is useful or selectively digesting a defined number of nucleotides from specific termini of a DNA or RNA molecule (single or double stranded) .

According to the present invention, the site-specific exonucleases or endonucleases of the present invention are generated by the construction of fusion proteins wherein the fusion is a hybrid between a nuclease and a ligand. The fusion is constructed in such a manner as to retain the catalytic activity of each of the components that make up the fused protein. In general, the fusion proteins of the present invention function in a similar manner; that is, the ligand component of the fusion protein dictates the position in which other catalytic activity of the fusion protein will act. Thus, the ligand binding activity has affinity for a "recognition sequence^M and once the recognition sequence is bound by the fusion protein, the nuclease portion of the fusion protein cleaves, or hydrolyseε within or near this recognition sequence. The "recognition sequence" as used with the fusion proteins of the present invention is defined as the specific sequence in close association with the bound receptor.

Although the mode of action for cleavage with the endonuclease fusion protein of the present invention is similar to the enzyme function of type II restriction endonucleases, one important distinction is evident. In the present invention, the ligand portion of the fusion protein recognizes and binds to the receptor bound to the recognition sequence, placing in close proximity the nuclease and the recognition sequence to be cleaved. In this manner, any recognition sequence which can be bound (covalently or non-covalently) to a receptor will bind the fusion protein of the present invention, resulting in cleavage within or near that particular recognition sequence.

Any DNA or RNA molecule or sequence may be cleaved according the present invention. The DNA or RNA substrate is only limited by the endonuclease portion of the fusion protein toward the substrate. For example, the fusion protein of the present invention will cleave double stranded DNA if a double stranded specific endonuclease is used to construct the fusion protein of the invention. In a similar manner, to cleave single stranded DNA or RNA according the present invention, the endonuclease portion of the fusion protein should be a single stranded DNA or RNA specific endonuclease, such as Staphylococcal nuclease. Exonuclease digestion of single or double stranded DNA or RNA molecules have been accomplished using known exonucleases. Controlled digestion with these exonucleases is possible, but only in a general manner to produce a population of molecules with varying lengths, i.e, hydrolysis is controlled by varying enzyme concentrations, time, temperature and/or enzymatic conditions. In accordance with the present invention, however, controlled site-specific digestion of single stranded or double stranded nucleic acid molecules are accomplished in a defined manner, shortening particular termini of nucleic acid molecule to a predetermined length.

In one embodiment, the fusion protein of the present invention is specifically bound to a receptor bound to the substrate nucleotide sequence, and exonuclease of the fusion protein is attached near the substrate termini to be hydrolyzed. Upon attachment of the fusion protein to this region of the nucleic acid molecules, the exonuclease begins to digest that particular termini. Digestion is completed when the fusion protein is removed from its nucleic acid substrat (s) .

The receptor, according to the present invention, is covalently or non-covalently bound to a particular sequence (herein termed "recognition sequence") . By binding the ligand portion of the fusion protein ±o the receptor-nucleic acid complex, also in either a covalent or non-covalent manner, the exonuclease is capable of hydrolyzing the nucleic acid termini located near the ligand attachments. Exonuclease digestion to the extent of removing the ligand has the effect of removing the enzyme from its substrate, stopping exonuclease digestion. In this manner, positioning the receptor near the termini determines the extent of digestion of a particular termini.

Thus, the fusion protein is attached to the nucleotide sequence through the receptor-ligand interaction, allowing the exonuclease to begin hydrolysis of that specific termini. When hydrolysis removes the nucleotide within the sequence which contains the receptor, the fusion protein is effectively remove from interacting with the nucleotide sequence, thereby inhibiting the hydrolysis of the specific termini. To facilitate the removal of the fusion protein from interacting with the nucleotide sequence, a continuous flow reaction may be preferably used rather than batch reaction. In batch reaction, the fusion protein is capable of non-specific hydrolysis or cleavage resulting from casual contacts rather than due to binding of the fusion protein to the receptor- substrate. The continuous flow system may be used for both the exonuclease or endonuclease fusions of the present invention and entails the use of columns, filters, gels, foams or the like to which is bound a fusion protein of the invention. Passing a substrate- receptor through the matrix which contains the fusion protein, allows cleavage or hydrolysis of the substrate in a site-specific manner. Because the system facilitates the removal of the digested product, non-specific interactions are prevented. .-

The fusion protein may be bound to the continuous flow matrix non-covalently, but is preferably bound to the matrix in a covalent manner. The fusion protein may be immobilized in any way that does not destroy the desired biological and catalytic activities of such protein, including immobilization of the fusion protein through a receptor-ligand interaction. For example, the fusion protein may contain two ligands to allow the fusion to stick to and immobilized on the matrix, as well as bind the receptor associated with the substrate.

Reaction conditions (time, temperature, pH, and salt concentration) under which the enzymes of the present invention are used will vary depending upon the nuclease and ligand components of the fusion protein, and the number of sites to be cleaved or number of nucleotides to be hydrolyzed. Typically, conditions used are those that are optimal for the natural unfused nuclease. Such conditions are well known in the art. Those of skill in the art may alter conditions to achieve optimal nuclease and optimal binding activities for each fusion protein. Well known nuclease and affinity assays may be used to determine optimal reaction conditions for each fusion protein. II. Fusion Protein Types

The fusion proteins of the present invention have two basic types of activities associated with them, nuclease and ligand binding activity. The fusion protein is constructed.in such a manner as to retain each activity of the macromolecule components from which the fusion protein is derived. Thus, at a minimum, the fusion protein of the present invention is composed of two macromolecules, one of which is associated with the nuclease activity while the other has binding activity associated with it.

Those of skill in the art will appreciate that a number of nuclease and/or receptor molecules may be functionally linked to generate a number of fusion protein "types", having multiple activity and thus multiple functions. For example, a fusion protein of the present invention may include a hybrid molecule composed of a endonuclease, exonuclease and two different ligand molecules, binding separate receptors within the same nucleic acid molecule. Contacting this multi-functional fusion protein with the receptor containing substrate would result in site directed hydrolysis of a particular termini and endonuclease cleavage within or near a preselected site within the substrate sequence. Other combinations of nuclease, receptor and/or antireceptors will be readily apparent to those of skill in the art depending on the substrate used and the desired result.

A. Nuclease

At least one component of the fusion proteins of the present invention has nuclease activity. Protein nucleases of the present invention include both exonuclease and endonucleases well known to those of skill in the art.

Any macromolecule exhibiting nuclease activity, protein or otherwise, may be used according to the present invention including, but not limi ed to, metal chelates, nonprotein enzymes (ribozymes) , and catalytically active proteins (enzymes) . Such metal chelates may include Fe-EDTA, Cu-phenathroline, Fe- porphyrins and Fe (II) bleomycin (Carter et al. , Proc. Natl. Acad. Sci. 87.:9373-9377 (1990) and Sigman et al.. Annu. Rev. Biochem. 5):207-236 (1990)). Preferably, protein nucleases are used to construct the fusion proteins of the present invention. However, one embodiment of the present invention uses non-protein enzymes such as ribozymes and their functional derivatives (Zaug, A.J. , et al.. Science 211:470-475 (1986); Zaug, A.J. , et al.. Nature 234:429-433 (1986); Zaug, A.J., et al.. Biochemistry 25:4478-4482 (1986); Been, M.D. , et al.. Science 239:1412-1416 (1988); and Doudna et al.. Nature

339_:519-522 (1989)).

Illustrative exonucleases to construct fusion proteins of the present invention include both RNA and DNA specific exonucleases such as deoxyribonuclease I, exonuclease III, exonucleases VII, lambda exonuclease, mung bean nuclease, nuclease Bal-31, nuclease PI, ribonuclease B. cereus. ribonuclease C13, ribonuclease H, ribonuclease Phy I, ribonuclease Phy M, ribonuclease Tl, ribonuclease T2, and ribonuclease U2. Endonucleases used in accordance with the present invention may include any nuclease known to those of skill in the art. Endonucleases which are heat stable and recognize only four nucleotides are preferred. Such endonucleases include Haelll (Middleton et al.. J.Virol. l :42-50 (1972); Tag"! (Sato et al.. Proc. Natl. Acad. Sci. 74:542-546 (1977) and Alul (Roberts et al.. J. Mol. Biol. 102:157-165 (1976)). The only requirement in constructing the fusion protein of the invention is that the endonuclease used be a nonspecific nuclease, i.e., in its natural unfused state the endonuclease will not recognize and bind to particular recognition sequences.

The endonuclease fusion proteins of the present invention are capable of digesting double stranded or single stranded RNA and DNA molecules so long as the appropriate endonuclease with substrate specificity towards the desired strandedness is included as a component of the fusion protein. The preferred endonuclease component of a fusion protein to cleave single stranded DNA at a particular site according to the present invention is Staphylococcal nuclease.

Single stranded RNA is preferably cleaved by a fusion protein which contains the ribonuclease RNase-S, ribozyme, or Staphylococcal nuclease. Preferably, double stranded DNA is cleaved by fusion proteins composed of either Staphylococcal nuclease, or Alul, Haelll or Taq^0-!, while double stranded RNA is digested with fusion proteins of RNase A (Shalongo et al.. Biochemistry 28:4820-4825 (1989)).

B. Ligand

The ligand component of the fusion protein of the present invention may be any macromolecule or portion thereof which can recognize and bind to its corresponding receptor. The only limitation in accordance with present invention is that the ligand component of the fusion protein can not include a nucleic acid molecule, RNA or DNA. Table 1 illustrates possible ligand:receptor combinations for use in the present invention. Preferably, the ligand portion of the ligand-nuclease fusion is a catalytically active protein having affinity for a receptor. In this way, the protein-protein fusion can be obtained by recombinant DNA techniques well known to those of ordinary skill in the art. However , the ligand may be a non-protein compound, bound to the nuclease portion of the fusion protein by well known chemical means .

Table 1

Biotin Avidin, streptavidin, etc.

Avidin , streptavidin , etc Biotin

Antigens Antibodies

Lec ins Glycoconjugates

Glycocon j ugates Lectins

Enzymes Substrates, cofactors, inhibitors, etc.

Cations Anions

Hydrophobic sites Hydrophobic groups

Receptors Hormones, effectors, toxins, etc.

Transport proteins Vitamins, amino acids, sugars, etc.

Membranes Liposomes

Catalytically active protein ligands used to orm a fusion protein include any protein which has affinity for a receptor. Such ligands include, but are not limited to, avidin, streptavidin, antibodies or antigens, enzymes, lectins, hormones, effectors, or toxins. When the protein ligand is an antibody, either the whole antibody or Fab fragment is used to bind its corresponding antigen receptor. Alternatively, the antigen or catalytically active fragment thereof is the ligand portion of the fusion protein of the present invention. The most preferred protein ligand used in accordance with the present invention is avidin or streptavidin, which will bind its corresponding receptor, biotin.• ^" The preferred non-protein ligand constituent of the fusion protein is biotin or its catalytically active variants or fragments. When biotin is utilized as a ligand, the receptor attached to the nucleic acid is avidin or streptavidin.

III. Isolation of Fusion Proteins

Isolation of each part of the fusion protein, i.e., nuclease and ligand, can be accomplished separately, followed by chemical fusion of each constituent in vitro to form the fusion protein of the present invention. In this case, substantially pure natural or recombinant proteins or non-proteins are fused by well known chemical means.

Preferably, the fusion protein is constructed in vivo and then separated by well known purification techniques. In this preferred embodiment, protein- protein fusion molecules are obtained by functionally linking the genes coding for the ligand and nuclease. In order to link these genes in this manner, the active site of each gene is to be linked in proper reading frame at the DNA level so that the protein translated from the "hybrid" gene will contain the activities or a portion of the activities of each gene.

A. Cloning the nuclease or ligand genes

Any of a variety of procedures may be used to clone the nuclease or ligand gene for construction of the fusion protein of the present invention. One such method entails analyzing a shuttle vector library of DNA inserts (derived from an nuclease or ligand expressing cell) for the presence of an insert which contains the nuclease or ligand gene. Such an analysis may be conducted by transfecting cells with the vector and then assaying for expression of the nuclease or ligand catalytic activity. The preferred method for cloning these genes entails determining the amino acid sequence of the nuclease or ligand molecule. To accomplish this task the nuclease or ligand protein may be purified and analyzed by automated sequencors. Alternatively, either molecule may be fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin or trypsin (Oike, Y. et al.. J. Biol. Chem. 257:9751-9758 (1982);

Liu, C. et al. _f Int. J. Pept. Protein Res. 21:209-215 (1983)). Although it is possible to determine the entire amino acid sequence of these proteins, it is preferable to determine the sequence of peptide fragments of these molecules. If the peptides are greater than 10 amino acids long, the sequence information is generally sufficient to permit one to clone a gene such as the gene for a particular nuclease or ligand. Once one or more suitable peptide fragments have been sequenced, the DNA sequences capable of encoding them are examined. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene. 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357). The peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon. Although occasionally such amino acid sequences may be encoded by only a single oligonucleotide, frequently the amino acid sequence can be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of the set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same nucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains a nucleotide sequence that is identical to the nucleotide sequence of this gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.

In a manner exactly analogous to that described above, one may employ an oligonucleotide (or set of oligonucleotides) which have a nucleotide sequence that is complementary to the oligonucleotide sequence or set of sequences that is capable of encoding the peptide fragment. A suitable oligonucleotide, or set of oligonucleotides which is capable of encoding a fragment of the nuclease or ligand gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified (using the above- described procedure) , synthesized, and hybridized, by means well known in the art, against a DNA or, a cDNA preparation depending upon the source of the gene. Typically, isolation of eukaryotic genes is done by screening a cDNA library, while a DNA library is used to isolate prokaryotic genes. Techniques of nucleic acid hybridization are disclosed by Maniatis, T. et al. , In: Molecular Cloning, a Laboratory Manual. Second Edition, Coldspring Harbor, NY (1989) , and by Haymes, B.D. et al.. In: Nucleic Acid Hybrization. a Practical Approach. IRL Press, Washington, DC (1985) , which references are herein incorporated by reference.

The source of DNA or cDNA used will preferably have been enriched for the desired sequences. Such enrichment can most easily be obtained from cDNA obtained by extracting RNA from cells cultured under conditions which induce nuclease or ligand synthesis.

Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for streptavadin (Argarana et al.. Nucleic Acids Research 14(4. :1871-1882 (1986), avidin (Kulomma et al.. J. Cell Biochem. SUPP. part 2:210 (1988), human hepatitis type B antibody (Hong et al.. Korean J. Biochem.. 18(1) :7-18 (1986)), human aldehyde dehydrogenases (Hsu, L.C. et al.. Proc. Natl. Acad. Sci. USA 82:3771-3775 (1985)), fibronectin (Suzuki, S. et al.. Eur. Mol. Biol. Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter, P. et al.. Proc. Natl. Acad. Sci. USA 8.2:7889-7893 (1985)), tissue-type plasminogen activator (Pennica, D. et all . Nature 301:214-221 (1983)) and human term placental alkaline phosphatase complementary DNA (Kam, W. et al.. Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985)).

A number of nuclease genes have been cloned using various techniques including the genes for extracellular nuclease of Serratia-Marcascens (Ball et al.. Gene 52:183-192 (1987)), 5' exonuclease of bacteriophage T5 (Kaliman et al.. FEBS Letters 195:61- 64 (1986)), and endonuclease of canine adenovirus type 2 (Spibey et al.. J. Gen. Virol. 70fl. :165-172 (1989)). Wilson, Gene 74:281-289 (1988), describes four techniques for isolating and cloning restriction endonuclease and modification methylase genes. All of the above mentioned references describing cloning of nuclease genes as well as cloning of the ligands (streptavidin, avidin and antibodies) are incorporated by reference herein. In a alternative way of cloning the nuclease or ligand gene, a library of expression vectors is prepared by cloning DNA or cDNA, from a cell capable of expressing nuclease or ligand into an expression vector. The library is then screened for members capable of expressing a protein which binds to anti- nuclease or anti-ligand antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as the nuclease or ligand, or fragments or variants thereof.

B. Fusing the nuclease and ligand genes

The cloned nuclease or ligand genes, obtained through the methods described above, may be functionally linked to form a "hybrid" nuclease-ligand fusion gene. The entire structural gene or portions containing the active site may be fused in any combination and any order. The only requirement is that the structural genes or structural gene fragment be functionally linked. The resulting hybrid gene may then be operably linked to an expression vector, and introduced into bacterial, or eukaryotic cells to produce the nuclease-ligand fusion proteins of the present invention. Techniques for constructing fusion proteins are well known to those of skill in the art. For example. Murphy (U.S. patent 4,675,382) describe recombinant DNA techniques to make hybrid proteins of diphtheria toxin and a polypeptide ligand such as a hormone. Fusion of gene or fusion of their fragments may be constructed in accordance with conventional techni¬ ques, including blunt-ended or stagger-ended termini for ligation, restriction digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Such manipulations are disclosed by Maniatis, T. et al.. supra. and are well known in the art.

Fusion proteins have been constructed by recombinant DNA techniques by fusing immunoglobulin genes to other genes including Staphylococcus nuclease, the mouse oncogene c-myc, and klenow fragment of E. coli DNA polymerase I (Neuberger et al. Nature 312:604-612 (1984); Neuberger et al.. Trends in Biochemical Science. 347-394 (September 1985)).

C. Expressing the nuclease-ligand fusion genes

DNA molecules composed of a nuclease gene functionally linked to a ligand gene or at least portions of these genes can be operably linked into an expression vector and introduced into a host cell to enable the expression of these proteins by that cell.

Two DNA sequences (such as a promoter region sequence and a desired fusion protein encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not

(1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired protein encoding gene sequence, or

(3) interfere with the ability of the desired protein gene sequence to be transcribed by the promoter region sequence. A DNA sequence encoding a nuclease-ligand fusion protein may be recombined with vector DNA in accordance with conventional techniques. The present invention encompasses the expression of the desired fusion proteins in either prokaryotic or eukaryotic cells. Eukaryotic hosts include yeast (especially Saccharomyces. , fungi (especially Aspergillus) , mammalian cells (such as, for example, human or primate cells) either in vivo_f or in tissue culture. Yeast and mammalian cells provide substantial advantages in that they can also carry out post- translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in these hosts.

Yeast recognize leader sequences on cloned mammalian gene products and secrete peptides bearing leader sequences (i.e., pre-peptides) . Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites.

Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CHO-K1, and their derivatives. For a mammalian host, several possible vector systems are available for the expression of the desired fusion protein. A wide variety of transcriptional and translational regu¬ latory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, etc., may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated. Of interest are regulatory signals which are temperature- sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.

The expression of the desired fusion protein in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al.. J. Mol.

APPI. Gen. 1:273-288 (1982)); the TK promoter of

Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C. , et al.. Nature

(London) 290:304-310 (1981)); the yeast ga!4 gene promoter (Johnston, S.A. , et al.. Proc. Nat1. Acad. Sci. USA^ 79:6971-6975 (1982); Silver, P. . , et al.. Proc. Natl. Acad. Sci. fUSAI 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the desired fusion protein does not contain any intervening codons which are capable of encoding a methionine (i.e. , AUG) . The presence of such codons results either in the forma¬ tion of a fusion protein (if the AUG codon is in the same reading frame as the desired fusion protein encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the desired fusion protein encoding sequence) . The expression of the hormone receptor molecules can also be accomplished in procaryotic cells. Preferred prokaryotic hosts include bacteria such as E. coli. Bacillus. Streptomvces. Pseudomonas, Salmonella. Serratia. etc. The most preferred prokaryotic host is E, coli. Bacterial hosts of particular interest include E. coli K12 strain HMS174 (F^", rec A, r~_Ki₂ ^m+κi₂ ^Ri^fR) ^and BL21 (F^", omp T, r^" _Bm~_B) and other enterobacteria (such as Salmonella typhimur- ium or Serratia marcescens) , and various-Pseudomonas species. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

To express the desired receptor molecule in a prokaryotic cell (such as, for example, E. coli. B. subtilis. Pseudomonas. Streptomyces. etc.), it is necessary to operably link the desired fusion protein encoding sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible) . Examples of constitutive promoters include the int promoter of bacteriophage λ, and the bla promoter of the b-lactamase gene of pBR322, etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (P_L and P_R) , the trp, recA, lacZ, lad, gal, and tac promoters of E. coli. the a-amylase (Ulmanen, I., et al.. J. Bacteriol. 162:176-182 (1985)), the s-28- specific promoters of B. subtilis (Gilman, M.Z., et al.. Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli. Academic Press, Inc., NY (1982)), and Streptomvces promoters (Ward, J.M., et al.. Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B.R. , (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream from the gene-encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold, L. , et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).

The desired receptor fusion protein encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the desired receptor molecule may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may complement an auxotrophy in the host (such as leu2. or ura3. which are common yeast auxotrophic markers) , biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. In a preferred embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells .of different species.

Any of a series of yeast gene expression systems can be utilized. Examples of such expression vectors include the yeast 2-micron circle, the expression plasmids YEP13, YCP and YRP, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D. , et al.. Miami Wntr. Symp. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomvces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R. , Cell 28.:203-204 (1982)) .

For a mammalian host, several possible vector systems are available for expression. One class of vectors utilize DNA elements which provide autonomously replicating extra-chromosomal plasmids, derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus. A second class of vectors relies upon the integration of the desired gene sequences into the host chromosome. Cells which have stably integrated the introduced DNA into their chromosomes may be selected by also introducing one or more markers which allow selection of host cells which contain the expression vector. The marker may provide for prototropy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. The cDNA expression vectors incorporating such elements include those described by Okayama, H. , Mol. Cell. Biol. 3.:2B0 (1983) , and others.

Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli such as, for example, pBR322, ColEl, pSClOl, pACYC 184, TΓVX. Such plasmids are, for example, disclosed by Maniatis, T. , et al. (In: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli. Academic Press, NY (1982) , pp. 307-329) . Suitable Streptomvces plasmids include pIJlOl (Kendall, K.J. , et al.. J. Bacteriol. 169:4177-4183 (1987)), and Streptomvces bacteriophages such as C31 (Chater, K.F., et al. , In: Sixth International Symposium on Actinomycetales Biology. Akademiai Kaido, Budapest, Hungary (1986) , pp. 45-54) . Pseudomonas plasmids are reviewed by John, J.F., et al. (Rev. Infect. Pis. 8:693-704 (1986)), and Izaki, K. (Jon. J. Bacteriol. 33:729-742 (1978)).

Once the vector or DNA sequence containing the constructs has been prepared for expression, the DNA constructs may be introduced into an appropriate host. Various techniques may be employed, such as protoplast fusion, calcium phosphate precipitation, electropora- tion or other conventional techniques. After the fusion, the cells are grown in media and screened for appropriate activities. Expression of the sequence results in the production of the fusion protein of the present invention.

D. Isolation of the nuclease-ligand fusion proteins

The fusion protein molecules of the invention may be isolated and purified from the above-described recombinant molecules in accordance with conventional methods, such as extraction, precipitation, chromato- graphy, affinity chromatography, electrophoresis, or the like. Preferably, affinity chromatography directed to the ligand component of the fusion protein is used to purify the fusion proteins of the present invention. Carriers containing immobilized forms of either biotin or avidin have been used for isolation purposes. For a review of avidin-biotin affinity chromatography, see Wilchek et al.. Analytical Biochemistry 171:1-32 (1988). Furthermore, a number of commercially available carrier-streptavidin matrixes are known.

According to the present invention, assays to detect the presence of the nuclease fusion protein can be used during conventional biochemical purification methods to determine the presence of these enzymes.

Nuclease fusion proteins of the present invention can be identified on the basis of cleavage of its nuclease substrate. As substrates, there can be used, for example, Adenovirus-2 (Ad-2) DNA. The substrate (DNA or RNA) , after exposure to the fusion protein, are separated electrophoretically in agarose gels in buffer systems conventional for separation of the digested products. substrates are visualized under ultraviolet light in the presence of ethidium bromide. IV. Incorporation of Receptor into the Nucleic Acid Substrate

A. Receptor

Table 1 supra illustrates possible ligand:receptor or receptor:ligand combinations for use in the present invention. According to the present invention, a receptor may include any macromolecule or portion thereof capable of having-a binding affinity to a particular ligand. Receptors which recognize a particular ligand naturally exists or can be prepared. Receptors in accordance with the present invention may include both protein and non- protein molecules. Illustrative receptors include, but are not limited, biotin, avidin, streptavidin, antigens, antibodies, lactins, glycoconjugates, enzymes, substrates, cofactors, inhibitors, cations, anions, hydrophobic sites, hydrophobic groups, hormones, effectors, toxins, transport proteins, vitamins, amino acids, sugars, membranes, and liposomes.

Preferably, the receptor is a catalytically active non-protein molecule having affinity for its corresponding ligand. The receptor used in the present invention may .be attached directly or indirectly to the nucleic acid to be digested or hydrolyzed. Indirect attachment is accomplished by binding the receptor either non-covalently or covalently to a second molecule; the second molecule designed to recognize and bind to a particular recognition sequence within the DNA or RNA molecule to be cleaved or hydrolyzed. Alternatively, the receptor may be bound directly (covalently or non-covalently) to the DNA or RNA molecule which is to be reacted with the fusion protein of the present invention. B. Attachment

1. Direct attachment Directly binding the receptor to the RNA or DNA molecule which is to be cleaved or hydrolyzed may be accomplished by well .known chemical or enzymatic means. Depending upon which receptor is to be linked to the substrate, different procedures well known to those of skill in the art will be used. Biotin, for example, as a receptor according to the present invention, can be attached to a substrate by a number of chemical and enzymatic techniques. For a review of biotin-nucleic acid interactions, see Wilchek et al. , Analytical Biochemistry 171:1-31 (1988), which is herein incorporated by reference. Nucleotide-containing-receptormolecules (many of which are available commercially) can be used to directly incorporated receptors into a double stranded DNA or RNA through standard nick translation techniques. In this case, nucleotides within the double stranded sequence can be replaced with nucleotide-containing-receptor molecules. Biotin has been introduced into the growing DNA strand through the use of a biotinylated nucleotide triphosphate in conjunction with nick translated techniques (Langer et al.. Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981). Furthermore, biotin-containing-compounds have been found to be suitable for direct introduction of the biotin moiety into DNA (Forster et al.. Nucleic Acids Res. 13.:745-761 (1985); and Reisfeld et al.. Biochem. Biophys. Res. Comm. 142:519-526 (1987).

Alternatively, strand polymerization techniques to synthesize single stranded DNA or RNA provide a means to incorporate nucleotide-containing-receptor molecules in the de novo strand. By altering the receptor labeled nucleotides in a reaction mixture. i.e., A-receptor, G-receptor, T-receptor or C- receptor, it is possible to selectively label the new strand with receptors. In this way, the newly synthesized strand can contain receptors only at the A, G, C, or T nucleotide position throughout the sequence. The use of. combinations of two or more receptor-nucleotides allows the incorporation of receptor-containing-nucleotides at a number of nucleotide sites within the sequence. By setting up four reactions in which different nucleotide-containing-receptors are used, the present invention provides for a method to sequence DNA or RNA molecules. To sequence DNA or RNA according the present invention, each of the four reaction tubes which have incorporated the receptor at the A, G, C, or T nucleotide sites are incubated separately with the ligand-nuclease. By varying reaction conditions such as time, temperature, and fusion protein concentration, a random population of digested molecules can be generated in each reaction tube. The scission products of all four reactions are then analyzed by gel electrophoresis according to standard sequencing techniques.

In order to visualize the products, the de novo DNA or RNA strand is labelled before or after the nuclease digestion. Labels used according the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non-radioactive isotopic labels, fluorescent labels, and chemiluminescent labels.

2. Indirect attachment

In addition to the direct attachment of receptor to a nucleic acid substrate, it is possible to indirectly attachment receptor molecules to the DNA or RNA sequence to be cleaved. This method is the most preferred. In this embodiment, the receptor is bound

(covalently or non-covalently) to a macromolecule.

Well known chemical and enzymatic techniques to bind protein and non-protein molecules are available. This macromolecule-receptor complex then mediates the linkage of the receptor to the substrate by recognizing and binding to the substrate. In this manner, the receptor is indirectly bound to the nucleic acid substrate. Thus, the receptor in accordance with the present invention may for -a substrate-macromolecule-receptor complex which will bind the fusion protein of the present invention.

The macromolecule used in the indirect binding of the receptor to the substrate may be any protein or non-protein molecule which has affinity for DNA or RNA. Preferably, the macromolecule has affinity for specific sequences within the substrate, although a non-specific DNA or RNA binder may be used to randomly digest the substrate. Preparation of receptor- macromolecules such as antibodies bound to biotin, or enzyme labeled antibodies is described by Harlow et al. _f In: Antibodies. A Laboratory Manual. Coldspring Harbor, NY (1988) .

Most preferably, the site-specific macromolecule is an RNA or DNA nucleotide probe that is bound to the substrate by hybridization. Thus, according to the present invention, single stranded DNA or RNA macromolecule-containing-receptor is hybridized to the single stranded substrate by standard hybridization techniques well known to those of skill in the art.

Construction of synthetic oligonucleotides to which biotin residues have been attached is disclosed by Chu et al.. DNA 4_:327-331 (1985). A number of studies discussing biotinylation of nucleotide sequences and their use in hybridization are reviewed by Wilchek et al.. Analytical Biochemistry 171:1-32 (1988). V. Uses

Sequence-specific cleaving or hydrolyzing agents of the present invention have applications in gene cloning, structural studies of nucleic acids, and chromosomal mapping. The endonuclease fusion proteins of the present invention can be used to selectively cleave a predetermined site within any nucleotide sequence, RNA or DNA. This is particularly important when considering the fact that the available Type II restriction endonucleases cannot be tailor made to cleave any predetermined sequence. Known restriction endonucleases can only recognize and cleave within or near a specific recognition sequence. Therefore, these limitations often frustrate research efforts. The ligand-exonuclease fusions of the present invention can remove any predefined number of nucleotides from the termini of the DNA or RNA substrate. Controlled digestion with known exonucleases is possible, but only in a general manner to produce a population of molecules with varying lengths, i.e, hydrolysis is controlled by varying enzyme concentrations, time, temperature and/or enzymatic conditions. Thus, before the present invention, it was not possible to specifically control the number of nucleotides removed during hydrolysis of DNA or RNA. In accordance with the present invention, however, controlled digestion of single stranded or double stranded nucleic acid molecules are accomplished in a defined manner, shortening particular termini of nucleic acid molecule to a predetermined length.

Defined removal of a certain number of nucleotides from the termini is necessary when unwanted sequences are present, i.e., lethal or non- functional promoter sequences. Controlled hydrolysis of the 5' termini would facilitate the removal of these sequences upstream of the ATG initiation codon, thereby allowing replacement of the natural controlling elements of a particular gene. The present invention also provides for a method to sequence DNA or RNA.molecules. The usefulness of this feature of the invention is self-evident.

In addition to the usefulness of the hybrid enzymes for genetic research, the present invention also provides an agent for diagnosing diseases of-a patient. For example, a number of diseases caused by viral infection result in the introduction of genetic material into the host. According to the present invention, fusion proteins can be tailored to recognize and cleave these unique heterologous sequences and thus in a uninfected host, no digestion will occur. Gel electrophoresis of DNA samples from the host can therefore be analyzed after reaction with the site-specific fusion protein to determine viral infection. A number of viruses including, but not limited to, papilloma virus, hepatitis A virus, hepatitis B virus, herpes simplex virus, human immunodeficiency virus, HTLV-III, HTLV-I, maize mosaic virus, tobacco mosaic virus, and bacteriophage can be detected in prokaryotic as well as eukaryotic host.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. Example 1

Construction of a Chimeric Protein of Staphylococcal Nuclease and Streptavidin

The streptavidin- or avidin-biotin complex is very useful as an extremely versatile, general mediator in a wide variety of bioanalytical applications due largely to the exceptionally high affinity (k_d=10~¹⁵ M) and consequently high stability of the non-covalent interaction between the two compounds (Ausubel, F.M. et al.. In: Current

Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York, (1987) . Many biotin-containing derivatives have been found suitable for direct introduction of the biotin moiety into nucleotide sequences and some of them are commercially available. Furthermore, biotinylated oligonucleotides are available which can be introduced into DNA or RNA molecules by standard nick translation or strand polymerization techniques. Once the DNA or RNA substrates (i.e., plasmid, chromosome, etc.) are labeled with the biotin receptor, the sites of receptor location serve as a specific target which can then be recognized and in turn interact with streptavidin- or avidin-containing fusion proteins. Figure 1 shows the construction of a hybrid gene which encodes a chimeric protein of Staphylococcal nuclease and streptavidin. pFOG301 contains a NUC gene which encodes Staphylococcal nuclease (Shortle, D., Gene 22.:181-189 (1983)) and pTSA-1 contains the gene which encodes streptavidin (Sano et al.. Proc. Natl. Acad. Sci. 82:142-146 (1990) and Argarana et al.. Nucleic Acids Research 14:1871-1882 (1986)).

Amplification of NUC gene and introduction of a Ndel restriction site at the 5-end and a Xbal site at the 3' end of the amplified NUC gene is accomplished by using the oligonucleotides 5'-TTAGAATTCCATATGACAGAA TACTTATTAAGT-3' (Sequence I.D. No. 1) and 5'-TTACAATGATCTAGATTGACC TGAATCAGCGTT-3' (Sequence I.D. No. 2) as the primers to perform PCR to amplify the pFOG301 plasmid. PCR is performed according to manufacturer's procedures (Perkin-Elmer) . Approximately thirty cycles of 95°C (1 min) , 45^βC (1 min) , 75°C (2 min) are performed. 5 μl out the 100 μl PCR reaction mixture is analyzed to determine the success of the PCR reaction. The remaining reaction mixture is loaded on 1% agarose gel and the PCR product is purified by Geneclean according to manufacturer's procedure. (BIO 101 Inc. La Jolla, CA)

Amplification of the gene encoding streptavidin and introduction of a Xbal site at 5'-end and a BamHI site at the 3'-end of the amplified gene is accom¬ plished by using oligonucleotides 5'-GCCCTCTAGAGAGGC- CGGCATCACCGGCACC-3' (Sequence I.D. No. 3) and 5'-CGCGAGGATCCCTGCTGAACGGCGTCGAGCGG-3' (Sequence I.D. No. 4) as primers to do PCR to amplify the streptavidin gene from pTSA-1. PCR is performed according to manufacturer's procedures (Perkin - Elmer) . The PCR product is purified by Geneclean as previously described. The PCR products of NUC gene and the streptavidin gene are then cloned into pET3a expression vector (Novagen) by digesting separately the amplified NUC gene with Xbal and Ndel at 37°C for 2 hrs, the amplified streptavidin gene with Xbal and BamHI at 37°C for 2 hrs and the pET3a plasmid with Ndel and BamHI at 37°C for 2 hrs. All the reactions are stopped by phenol/CHC1₃ extraction and followed by EtOH precipitation and a 70% EtOH wash. The digested NUC and streptavidin DNA are then lypolized. The digested pET3a DNA is lyophilized and then is dephosphoylated and cleaned up according to standard methods (Maniatis et al.. Molecular Cloning. A Laboratory Manual. Second Edition, Cold Spring Harbor (1989)). The digested NUC gene and streptavidin gene are then ligated into the digested and dephosphoylated pET3a expression vector by standard method (Maniatis, supra) . Aliquots of the ligation reaction mixture is used to transform the HMS174 cell line (F^", hsdR, recA, Rif^R: Campbell et al.. Proc. Natl. Acad. Sci. USA 215:2276-2280 (1978) by standard techniques. HMS174 is available from Novagen. Several clones are picked and grown overnight in LB media with ampicillin. Plasmid DNA is obtained from each culture and analyzed by Ndel/BamHI restriction digestion. The plasmid with correct insert size is selected, and the DNA is used for the expression of the fusion gene. This plasmid DNA is designated as pETNAS-100 (Fig. 1) .

Example 2

Expression and purification of the chimeric protein containing Staphylococcal nuclease and streptavidin

The structural gene of the chimeric protein (Fig.

1) is expressed in E. coli . The pENS-100 is used to transform the BL21 (DE3) (F^", ompT, hsdS, gal: Studier et al.. J. Mol. Biol. 189:113-130 (1986) and Grodgerg et al.. J. Bacteriol. 170:1245-1253 (1988)) according to standard method (Maniatis, supra.. BL21 is available from Novagen. BL21 (DE3) carrying the pETNS-100 plasmid is grown at 37°C with shaking in LB medium supplement with 0.4% glucose, and ampicillin at 50 μl/ml. When the A600 reaches 1.0, 100 mM IPTG is added to a final concentration of 0.4 mM to induce the T7RNA polymerase gene under control of the lacUV5 promoter. After induction, the cells are incubated at 37°C with shaking for another 2 hrs (Sano et al.. Proc. Natl. Acad. Sci. 87:142-146 (1990)).

All procedures for purifying the fusion protein are carried out at 4°C. The induced culture (100 ml) from above is harvested by centrifugation at 1600 xg for 10 min. The cell pellet is washed with 100 ml of 100 mM NaCl, ImM EDTA, 10 mM Tris.HCl, pH 8.0 and is centrifuged as above. The cells are suspended in 10 ml of 2 mM EDTA, 30 mM Tris. HCl, pH 8.0, containing 1 mg of egg white lyεozyme and 0.1% Triton X-100.

MgS0₄ (1.0M), DNase I(lmg/ml), and RNase A (lmg/ml) are added to a final concentration of 12 mM, 10 μg/ml, and 10 μg/ml, respectively, and the mixture is allowed to stand at room temperature for 15 min. The cell lysate is centrifuged at 39,000 xg for 15 min. at room temperature, and the precipitate is washed again with 10 mis of 2 mM EDTA, 30 mM Tris. HCl, pH 8.0, 0.1% Triton X-100 followed by centrifugation as above. The precipitate is dissolved in 5 ml of 6M guanidine hydrochloride (pH 1.5) with gentle stirring and is dialyzed against 0.2 M NaHC0₃ (pH 8.7) to remove guanidine HCl. The dialysate is centrifuged at 39,000 xg for 15 min. and the supernatant is used as the expressed fusion protein fraction. (Sano et al.. Proc. Natl. Acad. Sci. 87:142-146 (1990)). ^'

The chimeric protein is further purified by biotin-affinity column, as previously described (Wilchek, M. et al.. Anal. Biochem. 171:1-32 (1988)).

Example 3

Site-specific cleavage reaction of target DNA

The polynucleotide substrate (a) and (b) as shown in Figure 2 is prepared from circular single-stranded M13mp7 DNA. Single-stranded M13mp7 DNA is prepared by transforming E. coli TGI cells with RF M13mp7 DNA (Amersham) according to the method of Messing (Methods of Enzvmology 101:20 (1983)). To prepare substrate (a) (Figure 2) , the resulting closed circular single-stranded DNA is linearized with BamHI (Been &

Champoux, Methods of . Enzvmolgv 101:90 (1983)). Approximately 30 μg of circular DNA in 80 μl of buffer (0.2 M NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA) was heated for 2 min at 95°C and then for 5 min at 65^βC. After being cooled to room temperature, 3 μl of 0.2-M MgCl₂ and 6 μl (72 units) of BamHI are added to the mixture followed by incubation overnight at 37°C. The DNA is purified on a 0.8% agarose gel and dephosphorylated with calf intestinal phosphatase (BRL) (Maniatis et al.. 1982). The 5'-terminus is then labeled with ³²P and the 3'-terminus of the BamHI-linearized DNA is labeled by a modified method (Challberg & Englund, Methods of Enzvmology 65:39 1980; Been & Champous, 1983). Briefly, approximately 4 μg of BamHI-digested DNA in 50 μl of buffer containing 20 mM Tris-HCl (pH 7.4), 7 mM MgCl₂, 50 mM NaCl, 10 mM DTT, 0.05 mM dGTP, 0.25 μM [α-³²P]dATP (Amersham, 800 Ci/mmol) , and 10 units of Klenόw fragment of DNA polymerase I is incubated for 30 min at 11°C. The DNA is ethanol precipitated twice in order to remove unincorporated dATP.

A second substrate (b) (Figure 2) is prepared by anneal ing the o l igonucleotide , 5'-CCCCTC-AAATGCTTTAAACAGTTCAGAAAA-3' (Sequence I.D. No. 5) , to nucleotides 460-489 of the circular single-stranded DNA. The double-stranded region is then cleaved with Dral. Approximately 20 μg of circular DNA in 45 μl of a buffer containing 4 μM 30-nt oligonucleotide, 10 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 10 mM MgCl₂ is heated to 70°C and then slowly cooled to 37°C followed by the addition of 5 μl (200 units) of Dral. The action was incubated overnight at 37°C. The DNA was purified on a 0.8% agarose gel, dephosphorylated with calf intestinal phosphatase, and labeled at its 5'-terminus as described above.

Biotinylation of the binding site oligonucleotide 5'-CCCGCACAAGCCGCT-3' (Sequence I.D. No. 6) as shown in Figure 2 is prepared according to manufacturer's procedures, (Clon. Tech) . This biotinylated oligonucleotide is used to indirectly attach the streptavidin-nuclease fusion protein prepared in

Example 2 to the DNA substrate (a or b) to be cleaved.

The fusion protein is used to cleave the substrates (a or b) by preparing a reaction mixture (total volume 10 μl) containing approximately 12 nM

³ P-labeled substrate DNA, 14 nM biotinated oligonucleotide and 14 nM of nuclease-streptavidin chimera in 50 mM Tris-HCl (pH 7.0) , 50 mM NaCl, and 1 mM EDTA. This mixture is heated for 2 min. at 65^βC and then adjusted to the desired temperature. The reaction is initiated by the addition of l μl 100 mM CaCl₂ and is quenched by the addition of 10 μl formamide-dye mixture containing 10 mM ethylene glycόl bis (3-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) after a specific reaction time. The quenched reaction mixture is heated at 90°C for 3 min followed by cooling on ice. The resulting DNA fragments are analyzed by electrophoresis on 5% polyacrylamide gels containing 7 M area (1:20 cross-linking) or on 1% agarose gels, followed by autoradiography at -80°C with Kodak XAR5 X-ray film. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in recombinant DNA technology, enzymology and/or related fields are intended to be within the scope of the following claims.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

SEQUENCΞ LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Chang, Yie-Hwa Smith, John A

(ii) TITLE OF INVENTION: Universal Site-Specific Nucleases

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Sterne, Keβsler, Goldstein & Fox

(B) STREET: 1225 Connecticut Ave. NW Suite 300

(C) CITY: Washington

(D) STATE: D.C.

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(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Cimbala, Michele A

(B) REGISTRATION NUMBER: 33,851

(C) REFERENCE/DOCKET NUMBER: 0609.2710000

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 466-0800

(B) TELEFAX: (202) 833-8716

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TTAGAATTCC ATATGACAGA ATACTTATTA AGT 33

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TTACAATGAT CTAGATTGAC CTGAATCAGC GTT 33

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCCCTCTAGA GAGGCCGGCA TCACCGGCAC C 31

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: : CGCGAGGATC CCTGCTGAAC GGCGTCGAGC GG 32

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CCCCTCAAAT GCTTTAAACA GTTCAGAAAA 30

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CCCGCACAAG CCGCT 15

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: : ATCGTCGTCT GGTAAACGAG GG 22

Claims (30)

WHAT IS CLAIMED IS:

1. A site-specific endonuclease capable of selectively cleaving any nucleotide sequence comprising a fusion protein, wherein said fusion protein is a hybrid between a ligand and a endonuclease.

2. The site-specific endonuclease of claim 1, wherein said nucleotide sequence to be cleaved is RNA.

3. The site-specific endonuclease of claim 1, wherein said nucleotide sequence to be cleaved is DNA.

4. The site-specific endonuclease of claim 1, wherein said nuclease is avidin nuclease or a catalytically active fragment thereof.

5. The site-specific endonuclease of claim 1, wherein said ligand is streptavidin or a catalytically active fragment thereof.

6. The site-specific endonuclease of claim I, wherein said nuclease is Staphylococcal nuclease or a catalytically active fragment thereof.

7. The site-specific endonuclease of claim 1, wherein said fusion protein is a Staphylococcal nuclease-streptavidin conjugate.

8. A site-specific exonuclease capable of selectively hydrolyzing any nucleotide sequence comprising a fusion protein, wherein said fusion protein is a hybrid between a ligand and a exonuclease.

9. The site-specific exonuclease of claim 4, wherein said nucleotide sequence to be cleaved is RNA.

10. The site-specific exonuclease of claim 4, wherein said nucleotide sequence to be cleaved is DNA.

11. A method for selectively cleaving any nucleotide sequence at a predetermined site comprising: a. binding a receptor to said nucleotide sequence to be cleaved; b. contacting said receptor containing nucleotide sequence with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a endonuclease; and c. incubating the components of step (b) for a time sufficient to cleave the receptor containing nucleotide sequence within or near its recognition sequence.

12. The method of claim 11, wherein said fusion protein is a Staphylococcal nuclease-streptavidin conjugate.

13. The method of claim 12, wherein the receptor is biotin.

14. A method for selectively cleaving any nucleotide sequence at a predetermined site comprising: a. binding a receptor to a macromolecule, wherein said macromolecule is capable of binding to said nucleotide sequence; b. contacting said receptor containing macromolecule with said nucleotide sequence; c. contacting the components of step (b) with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a endonuclease; and d. incubating the components of step (c) for a time sufficient to cleave said sequence within or near its recognition sequence.

15. The method of claim 14, wherein said macromolecule is an oligonucleotide complementary to a portion of the nucleotide sequence to be cleaved—

16. The method of claim 15, wherein said receptor is a biotin.

17. The method of claim 16, wherein said fusion protein is a Staphylococcal nuclease-streptavidin conjugate.

18. A method for selectively cleaving any nucleotide sequence at a predetermined site comprising: a. binding a receptor to said nucleotide sequence to be cleaved; b. contacting said receptor containing nucleotide sequence with an antireceptor capable of binding to said receptor; c. contacting said receptor-antireceptor containing nucleotide sequence with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a endonuclease; and d. incubating the components of step (c) for a time sufficient to cleave the receptor- antireceptor containing nucleotide sequence within or near its recognition sequence.

19. A method to hydrolyze any nucleotide sequence by removing a defined number of nucleotides from the termini of said sequence comprising: a. binding a receptor to said nucleotide sequence to be hydrolyzed; b. contacting said receptor containing nucleotide sequence with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a exonuclease; and c. incubating the components of step (b) for a time sufficient to hydrolyze the receptor containing nucleotide sequence.

20. A method to hydrolyze any nucleotide sequence by removing a defined number of nucleotides from the termini of said sequence comprising: a. binding a receptor to a macromolecule, wherein said macromolecule is capable of binding to said nucleotide sequence; b. contacting said receptor containing macromolecule with said nucleotide sequence; c. contacting the components of step (b) with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a exonuclease; and d. incubating the components of step (c) for a time sufficient to hydrolyze said sequence.

21. A method to hydrolyze any nucleotide sequence by removing a defined number of nucleotides from the termini of said sequence comprising: a. binding a receptor to said nucleotide sequence to be hydrolyzed; b. contacting said receptor containing nucleotide sequence with an antireceptor capable of binding to said receptor; c. contacting said receptor-antireceptor containing nucleotide sequence with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a exonuclease; and d. incubating the components of step (c) for a time sufficient to hydrolyze the receptor- antireceptor containing nucleotide sequence.

22. A method of producing a DNA or RNA fragment comprising: a. binding a receptor to a DNA or RNA molecule to be cleaved; b. contacting said receptor containing DNA or RNA with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a endonuclease; c. incubating the components of step (b) for a time sufficient to cleave the receptor containing DNA or RNA. d. obtaining the cleaved DNA or RNA fragments from step (c) .

23. A method of producing a DNA or RNA fragment comprising: a. binding a receptor to a macromolecule, wherein said macromolecule is capable of binding to said nucleotide sequence; b. contacting said receptor containing macromolecule with a DNA or RNA molecule to be cleaved; c. contacting the components of step (b) with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a endonuclease; d. incubating the components of step (c) for a time sufficient to cleave the said DNA or RNA molecule; and e. obtaining the cleaved DNA or RNA fragments from step (d) .

24. The method of claim 23, wherein said macromolecule is an oligonucleotide complementary to a portion of the nucleotide sequence to be cleaved.

25. The method of claim 24, wherein said receptor is a biotin.

26. The method of claim 25, wherein said fusion protein is a Staphylococcal nuclease-streptavidin conjugate.

27. A method of producing a DNA or RNA fragment comprising: a. binding a receptor to a DNA or RNA molecule to be cleaved; b. contacting saidreceptor containing DNA or RNA with an antireceptor capable of binding to said receptor; c. contacting said receptor-antireceptor containing DNA or RNA with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a endonuclease; d. incubating the components of step (c) for a time sufficient to cleave the receptor- antireceptor containing DNA or RNA; and e. obtaining the cleaved DNA or RNA fragments from step (d) .

28. A method of sequencing DNA or RNA comprising: a. binding a receptor to a specific class of nucleotides. A, G, C, or T, throughout the DNA or RNA sequence; b. contacting said receptor containing nucleotide sequence with a fusion protein, wherein said fusion protein is a hybrid between a ligand and a endonuclease; c. incubating the components of step (b) for a time sufficient to generate a random population of cleavage products; and d. comparing the cleavage products for each class of nucleotides by separating said cleavage products by gel electrophoresis.

29. The method of claim 28, wherein said receptor is a biotin.

30. The method of claim 29, wherein said fusion protein is a Staphylococcal nuclease-streptavidin conjugate.