DNA transformation efficiency by inhibiting host cell restriction Number:7,101,713 from the United States Patent and Trademark Office (PTO) owispatent

Title: DNA transformation efficiency by inhibiting host cell restriction

Abstract: It can be difficult to achieve efficient transformation of many strains of bacterial cells due in part to the presence of one or more restriction and modification (R-M) systems in the cells that restricts unmodified transforming DNA. Phage T7 OCR protein is a potent inhibitor of Type I R-M systems. Methods are disclosed for improving transformation efficiency of Eubacterial and Archaebacterial cells having an R-M system by introducing into the cells an inhibitor of the restriction activity. For example, addition of 1 5 micrograms of T7 OCR protein to 50 microliters of electrocompetent cells having a Type I R-M system prior to electroporation significantly increased transformation efficiency by unmodified plasmids, fosmid clones, and artificial transposons comprising synaptic complexes. Other inhibitors or restriction activity, such as phage-encoded proteins and proteins encoded by conjugative plasmids, as well as other disclosed inhibitors, also can be used to improve transformation (and transposition) efficiency. Host cells with heritable improved transformation efficiency can be made by transforming a cell having an R-M system with an expressible gene which encodes an inhibitor of the restriction activity, which gene can be conditionally expressible. Transient expression of a gene on an unmodified transforming DNA can also be improved by in vivo inhibition of restriction in host cells having an R-M system. Kits and compositions for carrying out the methods of the invention and host cells made using the methods are also disclosed.

Patent Number: 7,101,713 Issued on 09/05/2006 to Hoffman,   et al.

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Inventors:	Hoffman; Leslie M. (Madison, WI), Jendrisak; Jerome J. (Madison, WI)
Assignee:	Epicentre Technologies Corporation (Madison, WI)
Appl. No.:	10/147,564
Filed:	May 16, 2002

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Current U.S. Class:	435/473 ; 435/474; 435/476
Current International Class:	C12N 15/64 (20060101)
Field of Search:	435/471,472,473,475,476,477,478,479,483,484,485,486,487,488 530/300,350

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References Cited [Referenced By]

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U.S. Patent Documents

	4910140		March 1990		Dower
	6294385		September 2001		Goryshin et al.

Other References

Reuter et al., Zeitschrift Fur All gemeine Mikrobiologie 20 (5): 345-354, 1980. cited by examiner . Krueger et al., Mol. Gen. Genet. 185: 457-461, 1982. cited by examiner . Mark et al., J. Biol. Chem. 256: 2573-2578, 1981. cited by examiner.

Primary Examiner: Vogel; Nancy
Attorney, Agent or Firm: Quarles & Brady LLP

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Claims

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What is claimed is:

1. A method for improving transformation efficiency of a prokaryotic host cell having a Type I restriction and modification (R-M) system by an unmodified transforming DNA, said method comprising introducing into the prokaryotic host cell an isolated OCR protein, which protein is encoded by a T3 or T7 bacteriophage gene, together with the unmodified transforming DNA.

2. The method of claim 1 wherein the prokaryotic host cell is selected from the group consisting of Eubacteria and Archaebacteria.

3. The method of claim 1 wherein the OCR protein is encoded by the 0.3 ocr gene of bacteriophage T7 or bacteriophage T3.

4. The method of claim 1 wherein the OCR protein and the unmodified transforming DNA are introduced into the prokaryotic host cell by electroporation.

5. The method of claim 1 wherein the unmodified transforming DNA is a polynucleotide comprising recombinant DNA in a vector.

6. The method of claim 1 wherein the unmodified transforming DNA is selected from the group consisting of a transposon and an artificial transposon.

7. The method of claim 1 wherein the unmodified transforming DNA is a polynucleotide selected from the group consisting of a plasmid, a suicide plasmid, a shuttle vector, a cosmid, a fosmid, an oligonucleotide, an amplicon, an episome, a BAC, a YAC, and a replicon.

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Description

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BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and compositions for improving DNA transformation efficiency of host strains by inhibiting host cell restriction activity.

2. Prior Art

DNA transformation is an important process in molecular biology. Transformation is the process of contacting a cell with a DNA molecule under conditions so that the DNA is taken up and incorporated into the cell so has to produce a heritable genotype. The cell into which the DNA is introduced is commonly referred to a "host cell." Host cells used for DNA transformation include, but are not limited to, bacterial cells. A host cell can be a cell comprising any organism. Many aspects of transformation, including some methods and information for different types of host cells, have been described in a review [Smith, H. O., et al., "Genetic Transformation," In: Ann. Rev. Biochem., 50: 41 68, 1981), incorporated herein by reference.

One purpose for transformation, the process of which is also referred to as "transforming a host cell," is to clone a DNA molecule comprising natural or synthetic DNA. In order to clone a DNA molecule, the DNA is joined to a replicable vector, such as a plasmid or bacteriophage vector, which is capable of replicating autonomously in the host cell. Once the DNA is joined to the vector, the resulting "recombinant DNA" is used to transform a suitable host cell. Then the host cell is grown in a culture medium in order to multiply the number of cells, which are descendents of the host cell, each of which contains one or more copies of the recombinant DNA. This process of "DNA cloning" or "molecular cloning" thus enables one to obtain multiple copies of a single recombinant DNA molecule. Many cloning vectors and methods related to cloning and transformation are described or cited by Joseph Sambrook and David W. Russell [Molecular Cloning--A Laboratory Manual Volumes 1 3, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001], incorporated herein by reference.

To maintain the plasmid vector in the cell, the vector DNA typically contains a selectable marker. Any selectable marker known in the art can be used. For an E. coli vector, any gene that conveys resistance to any antibiotic effective in the cell, or any gene that conveys a readily identifiable or selectable phenotypic change can be used. Preferably, antibiotic-resistance markers, including, but not limited to, genes that confer resistance to kanamycin or other aminoglycoside antibiotics such as dihydrostreptomycin, gentamycin, neomycin, paromycin, or streptomycin, or to amphenicols, such as chloramphenicol (for which the gene is known as chloramphenicol transacetylase or CAT), are used. Those with skill in the art will know or know how to find other antibiotic-resistance genes and non-antibiotic selection methods to select for maintenance of a plasmid or other vector. A host cell can also be transformed by natural or synthetic DNA which is not joined to (or "cloned in") a replicable vector. For example, it is known in the art that if at least a portion of the transforming DNA has homology with DNA in the host cell, the transforming DNA can be incorporated into a host cell that has a "homologous recombination system" [e.g.s, See: Camerini, R. D., and Hsieh, P., "Homologous recombination proteins in prokaryotes and eukaryotes," Annual Rev. Genetics, 29: 509 552, 1995; Kowalczykowski, S. C., et al., "Biochemistry of homologous recombination in Escherichia coli," Microbiol. Rev., 58: 401 465, 1994; Heyer, W. D., and Kohli, J., "Homologous recombination," Experientia, 50: 189 191, 1994; Smith, G. R., "Homologous recombination in prokaryotes," Microbiol. Rev., 52: 1 28, 1988]. A homologous recombination system is comprised of enzymes which catalyze various reactions so as to bring about incorporation of foreign DNA which has at least some homology to DNA in the host cell into the DNA of the host cell.

Recently, researchers demonstrated that a host cell also can be transformed with a synaptic complex consisting of a DNA molecule comprising an artificial transposon and a transposase enzyme which recognizes the ends of the transposon [Goryshin and Reznikoff, U.S. Pat. No. 6,294,385]. Following transformation of the host cell with the synaptic complex, the transposase in the complex is activated by intracellular magnesium ions in the host cell so as to catalyze transposition of the transposon into the cellular DNA of the transformed host cell. In this case, the transforming DNA is introduced into the host cell's DNA and therefore, the transforming DNA does not require the capability for autonomous replication in the host cell.

A variety of methods and conditions are known in the art for improving the efficiency of transformation of a given host cell. These methods and conditions vary for different organisms and even for different strains of a particular organism. The efficiency of transformation is usually measured by stating the number of independently-transformed host cells that are obtained using a certain quantity of a transforming DNA under defined conditions of transformation. In most cases in which the transformation efficiency is determined, the transforming DNA encodes at least one gene, such as an antibiotic resistance gene, which can be used as a selectable marker. It is commonly stated in the art, for example, that 10.sup.2. up to about 10.sup.10. colony forming units (cfu) of a transformed Escherichia coli host strain (which is resistant to the antibiotic in the medium because of the presence of the antibiotic-resistance gene in the transforming DNA) are obtained per microgram of transforming DNA under the specified conditions, which would be described. In most experiments in which the transformation efficiency is determined using transforming DNA that encodes an antibiotic resistance gene, equal quantities of host cells which have been treated with transforming DNA are plated on two different media; one medium contains the antibiotic for which the transforming DNA encodes resistance and the other medium does not contain this antibiotic. Thus, the number of colonies which grow on the antibiotic-containing medium is used to calculate the cfu per quantity of transforming DNA under the conditions used, and the number of cfu on the antibiotic-containing plates divided by the number of cfu on the medium without antibiotic indicates the relative proportion of the host cells that were transformed by the transforming DNA.

One method to increase the uptake of a DNA molecule, or the efficiency of transformation, is to treat the host cell so as to increase its ability (or "competence") to take up DNA under transformation conditions. For example, treatment of bacterial cells with calcium chloride increases the cells' ability to take up DNA (Mandel, M. and Higa, A., "Calcium-dependent bacteriphage DNA infection," J. Mol. Biol., 53: 159 162, 1970; Cohen, S. N. et al., "Construction of biologically functional bacterial plasmids in vitro," Proc. Natl. Acad. Sci. USA, 73: 3240 3244, 1973). Many other variations have been described for increasing the competence of the cells by treating them with chemicals (e.g., Hanahan, D. "Studies on transformation of Escherichia coli with plasmids," Gene, 24: 317 326, 1983; Hanahan, D., et al., "Plasmid transformation of E. coli and other bacteria," Methods, Enzymol. 204: 63 113, 1991). This process is commonly referred to in the art as "making the cells chemically competent". Alternatively, a preferred method of transformation employs pulses of electric current to introduce the DNA into host cells, a process referred to as electroporation. The cells used for electroporation are usually grown and treated so as to make them more capable of transformation by electroporation and such cells are referred to in the art as "electrocompetent cells". Electroporation of prokaryotic cells with DNA material is typically achieved by the subjecting host cells with high-intensity electric pulses to make the cell walls "porous", as described by U.S. Pat. No. 4,910,140. Other methods for transformation of host cells are known by those with skill in the art, such as, but not limited to, introducing transforming DNA into the host cells by means of microinjection. or by encapsidation in a bacteriophage or viral coat, or in a liposome.

Transformation of various types of cells, including different strains of bacterial cells, can be difficult to achieve or very inefficient. The various factors that influence the efficiency of transformation are poorly understood. In order to study and manipulate various types of cells, it is therefore critical to find methods to increase the efficiency of DNA transformation of these cells.

The presence of a restriction and modification system ("R-M system") in a host cell is well known in the art to be an important factor in preventing or reducing the efficiency of transformation by transforming DNA that is unmodified with respect to the R-M system in the host cell. Type I, Type II, and Type III R-M systems are described and discussed and citations are given in review articles by Noreen E. Murray ["Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertany and Weigle)." Microbiol. Molec. Biol. Rev., 64: 412 434, 2000; "Immigration control of DNA in bacteria: self versus non-self," Microbiology, 148: 3 20, 2002] and Robert Yuan ["Structure and Mechanism of Multifunctional Restriction Endonucleases," Ann. Rev. Biochem., 50: 285 315, 1981], and in the 3.sup.rd edition of Sambrook and Russell's Molecular Cloning--A Laboratory Manual, all of which are incorporated herein by reference. Thus, as discussed by Murray [op cif], the DNA in a prokaryotic host cell that has an R-M system is modified so that the cell's own DNA is protected from digestion (or "restriction") by an endonuclease (also referred to as a "restriction enzyme") that is present in the cell, but transforming DNA from another cell which lacks the modification system is not protected from digestion by the endonuclease (if the transforming DNA has one or more sequences which are recognized by the endonuclease as sites for digestion by the endonuclease). The modification enzymes that protect the DNA from digestion are generally DNA methyltransferases which catalyze methylation of specific nucleic acid bases within the target sequence that is digested by the endonuclease. Thus, the modification enzymes block endonuclease digestion of the methylated (or "modified") DNA. As stated by Murray [Microbiol. Molec. Biol. Rev., 64: p. 413, 2000], "Classically, a restriction enzyme is accompanied by it's cognate modification enzyme, and the two comprise a restriction and modification (R-M) system. Most restriction systems conform to this classical pattern. There are, however, some restriction endonucleases that attack DNA only when their target sequence is modified; such modification-dependent restriction enzymes do not, therefore, coexist with a cognate modification enzyme . . . The classical R-M systems and the modification-dependent restriction enzymes share the potential to attack DNA derived from different strains and thereby restrict DNA transfer."

DNA which lacks methyl groups or other modifications which block digestion by the restriction activity portion of an R-M system is referred to as "unmodified" DNA with respect to that R-M system. Unmodified DNA molecules introduced into host cells are often attacked by R-M systems that recognize specific sequences in the DNA and rapidly cleave the DNA into fragments. For example, Type I R-M enzymes are thought to exist in a high percentage of Eubacteria and Archaebacteria, and pose a barrier for entry and establishment of foreign DNA [Murray, N. E., ibid; and Titherage, A. J. B., et al., Nucl. Acids Res., 29: 4195 4205, 2001]. There are many situations in which the modification state of transforming DNA leads to restriction in the recipient host cell [Ando, T. et al., Molec. Microbiol., 37: 1052 1065, 2000]. It would be highly desirable to improve transformation efficiency of such strains having R-M systems.

Many R-M systems are encoded by multiple genes. By way of example, but not of limitation, Type I R-M systems are generally encoded by three genes hsdR, hsdM, and hsdS--which encode three protein subunits, colloquially referred to as "R" (for "restriction"), "M" (for "modification"), and "S" (for "specificity"). These subunits can exist in two kinds of functional complexes in bacteria. An R-M complex with all three subunits (R.sub.2., M.sub.2., S.sub.1.) comprises an R-M system. Another complex, which has only methyltransferase activity and no restriction activity, comprises only two kinds of subunits (M.sub.2., S.sub.1.) and lacks R subunits [See: Murray, N. E., Microbiology, 148: 3 20, 2002]. Type III R-M system have similarities to Type I systems with respect to their molecular structure and other characteristics, whereas Type II R-M systems have separate restriction enzymes (which are the common commercial enzymes used in molecular biology) and methyltransferase modification enzymes [ibid].

Because R-M systems present a barrier to transformation of unmodified DNA, special bacterial strains have been developed for a limited number of organisms which are devoid of the known R-M systems for use in cloning and other applications in which unmodified DNA is used for transformation. Such strains are commonly referred to in the art as being "restriction minus" and the specific genotype of a strain is often presented so as to indicate which R-M system has been deleted or inactivated by mutation in the strain. These restriction minus strains are used as host cells because they do not digest or restrict the unmodified transforming DNA that enters the cells during transformation. By way of example, but not of limitation, an electrocompetent form of an E. coli strain for use in cloning when electroporation is used for transformation is commercially available under the trademarked name of E. coli TransforMax.sup.TM. EC100.sup.TM. (EPICENTRE); this strain is restriction minus and the genotype with respect to the R-M systems is described as "mcrA delta(mrr--hsdRMS--mcrBC)." Since E. coli strain TransforMax.sup.TM. EC10.sup.TM. is restriction minus, and also because the cells have been prepared so as to maximize the electrocompetence of the cell, very high transformation efficiencies are obtained when unmodified transforming DNA is used to transform the cells by electroporation. On the other hand, if unmodified DNA molecules, including, for example, plasmids or other replicons, or transposons, are taken from non-modifying cells or prepared synthetically or by PCR or by other means, and then used to transform competent cells which have an R-M system, such as, for example, cells having a Type I R-M system, the transformation will either be impossible or the transformation efficiency will be greatly reduced. Since it is highly desirable to be able to study and to be able to transform many cells which have an R-M system, including many pathogens and other strains of medical or commercial interest, there is a great need in the art to develop methods for improving transformation of such cells with unmodified DNA. It is therefore highly desirable in the art to find methods to overcome the barrier to transformation of host cells which have an R-M system which digests unmodified transforming DNA.

Many examples are known in which the R-M system barrier to transformation by unmodified phage and conjugative plasmid DNA is overcome by natural means. By way of example, but not of limitation, bacteriophages T7 and T3, which infect enteric bacterial cells, have evolved natural mechanisms to defeat host R-M systems. These bacteriophages produce a polypeptide encoded by the 0.3 ocr gene early in infection, before the remainder of the phage genome is internalized in the host cell, that mimics a DNA molecule; this polypeptide binds to the R-M system and prevents it from binding to DNA substrates, thereby inhibiting restriction activity of the R-M system [Bandyopadhhay, P. K., et al., J. Mol. Biol., 182: 567 578, 1985]. It has been shown that this polypeptide, referred to as "Overcomes Classical Restriction" or "OCR," has a conformation resembling the bent structure of unmodified DNA when it binds to Type I R-M complexes, and that OCR binds the restriction complexes tightly [Atanasiu, O., et al., Nucl. Acids Res., 29: 3059 3068,12, 2001 and Walkinshaw, M. D., et al., Molec. Cell, 9: 187 194, 2002]. Thus, by acting as a molecular "decoy", OCR protein inhibits restriction and prevents destruction of entering bacteriophage DNA.

In addition to phage-encoded inhibitors of restriction activity like the OCR proteins, inhibitors of the restriction activity of R-M systems have also been identified in bacteria which contain naturally-occurring transmissible plasmids, meaning plasmids which can be transferred from one bacterial strain to another by conjugation. By way of example, but not of limitation, it has been reported that certain transmissible plasmids have genes designated ardA, or ardB, or ardC that encode polypeptides called "Ard" for "alleviation of restriction of DNA" which provide general protection against restriction by all Type I R-M systems [Belogurov, A. A., et al, "IncN plamid pKM101 and Incl1 plasmid Col1b-P9 encode homologous anti-restriction proteins in their leading regions," J. Bacteriol., 174: 5079 5085, 1992; Belogurov, A. A., et al., "Plasmid pKM101 encodes two non-homologous anti-restriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences," J. Bacteriol., 175: 4843 4850, 1993; Belogurov, A. A., et al., "Antirestriction protein Ard (Type C) encoded by IncW plasmid pSa has a high similarity to the `protein transport` domain of TraC1 primase of promiscuous plasmid RP4," J. Mol. Biol., 296: 969 977, 2000]. Belogurov et al. reported that, in addition to inhibiting Type I R-M systems, ArdA also inhibits the Type II enzyme EcoRI. Recently, it was reported that, as with T3- and T7-encoded OCR Protein, the ard gene is in the leading edge of the DNA that is transferred into the host cell during conjugation, and, by a using special promoters within secondary structures of single-stranded DNA, it is transcribed into Ard protein prior to entrance of the remaining plasmid DNA into the host cell [Althorpe, N. J., et al., "Transient transcriptional activation of the Incl1 plasmid anti-restriction gene (ardA) and SOS inhibition gene (psiB) early in conjugation," Mol. Microbiol. 31: 133 142, 1999; Bates, S. R., et al., "Expression of leading region genes on Incl1 plasmid Col1b-P9: genetic evidence for single-stranded DNA transcription," Microbiology, 145: 2655 2662, 1999]. It has been proposed that Ard Proteins alleviate restriction of DNA by mimicking the sequences of the specificity ("S") subunits of Type I R-M systems and displace this S subunit from the R-M complex [Belogurov, A. A., and Delver, E. P., "A motif conserved among the type I restriction-modification enzymes and antirestriction proteins: a possible basis for mechanism of action fo plasmid-encoded antirestriction functions," Nucleic Acids Res., 23: 785 787, 1995], but it is also possible that the mechanism is similar to that of OCR Protein.

Other natural mechanisms by which unmodified forms of phages, plasmids and at least one conjugative transposon avoid restriction in host cells having an R-M system in which they would otherwise be degraded are described, discussed and referenced [Murray, N. E., Microbiol. Molec. Biol. Rev., 64: 412 434, 2000 (especially the section entitled "Mechanisms by Which Plasmids and Phages Avoid Restriction" on pp. 425 426); Bickle, T. A. and Kruger, D. H., "Biology of DNA restriction," Microbiol. Rev. 57: 434 450, 1993; and Kruger, D. H. and Bickle, T. A. "Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts," Microbiol. Rev. 47: 345 360,1983], all of which are incorporated herein by reference.

Although it is known in the art that one can increase the transformation efficiency of transforming DNA for a host cell having an R-M system by modifying the restriction sites on the transforming DNA prior to transformation, this is not practical in many cases. For example, one might not know what restriction sites are present on the DNA, what sites on the DNA must be modified to protect it, or how to achieve the desired modification. Also, although some of the R-M systems that are present in a number of E. coli strains have been identified, this is not the case for many other strains of E. coli and definitely is not the case for most other strains of eubacteria and archaebacteria. Noreen Murray has discussed the detection, distribution and diversity of R-M systems [Microbiol. Molec. Biol. Rev., 64: 412 434, 2000 (especially, see pp. 426 430)], incorporated herein by reference. As discussed therein, at least about half of the bacteria in a sequence database had genes that appeared to be Type I-specific R-M genes. More than 1% of the small genome of the medically important bacterium Helicobacter pylori encoded R-M systems, three of which were Type I [Tomb, J. F., et al., "The complete genome sequence of the gastric pathogen Helicobacter pylori," Nature, 388: 539 547, 1997]. It would be difficult to overcome the complex R-M systems of H. pylori by modifying a transforming DNA in order to avoid its restriction by such a host cell. The R-M systems present in most other bacteria of medical, commercial and food importance are not yet defined. Even more challenging is the fact that the actual restriction sites on the DNA of Type I and Type III R-M systems are very difficult to define and identify even after the presence of a R-M system has been shown to be present by screening for R-M genes. Further, the modification enzymes comprising R-M systems from many organisms have not been purified, making it difficult to modify transforming DNA using in vitro treatment with modifying enzymes.

It would be highly desirable to be able to increase the transformation efficiency of many host cells in which the R-M systems have not yet been defined, even if the sequence of the transforming DNA is not known. The present invention provides new and powerful methods for achieving transformation and increasing the transformation efficiency by unmodified DNA for many host cells having an R-M system. These methods do not require prior knowledge of the restriction sites on the unmodified transforming DNA or the restriction specificity of the R-M system. They can be used to increase transformation efficiency of a variety of host cells having R-M systems which restrict unmodified transforming DNA at different sites. The present invention also provides methods for genetically modifying host cells which have an R-M system so they can be transformed by unmodified transforming DNA with higher efficiency. The invention also discloses kits and compositions for carrying out the methods of the invention, or which are made using these methods.

OBJECTS OF THE INVENTION

It is currently very difficult or impossible to transform cells of many organisms with unmodified DNA, and even when transformation is possible for a particular kind of cell, the efficiency is usually very low. It is an object of the invention to provide a method for improving transformation efficiency of host cells having a restriction and modification (R-M) system with an unmodified transforming DNA by introducing into the host cell an inhibitor of the restriction activity of the host cell's R-M system so as to reduce or block restriction of the unmodified transforming DNA during transformation. A preferred object of the invention is to provide methods to improve transformation efficiency of host cells selected from among Eubacteria or Archaebacteria. Another preferred object of the invention is to provide methods to improve transformation efficiency of Eubacterial and Archaebacterial host cells having at least one R-M system selected from among a Type I R-M system, a Type II R-M system, or a Type III R-M system by unmodified transforming DNA that has at least one site which, in the absence of an inhibitor for the restriction activity of the host cell, would be restricted in the host cell.

A primary object of the invention is to provide methods to improve transformation efficiency of host cells by unmodified DNA by introducing into the host cells a composition or compositions that bind to a site on an enzyme of the host cells' R-M system so as to reduce or block restriction of unmodified transforming DNA during transformation. Most preferably, the composition will inhibit the restriction activity of a broad range of Eubacterial and Archaebacterial cells.

Since electroporation is the most efficient method for transforming some kinds of Eubacterial or Archaebacterial cells, an especially preferred object of the invention is to provide methods for improving transformation efficiency of host cells with unmodified transforming DNA by electroporation, wherein a composition that binds to a site on an enzyme of the host cells' R-M system is introduced into the host cells during the process of electroporation so as to reduce or block restriction of the unmodified transforming DNA.

It is an object of this invention to improve the stability of foreign genetic material delivered into host cells in molecular cloning and recombinant DNA applications. A preferred object of the invention is to improve transformation efficiency of a host cell having an R-M system with unmodified transforming DNA comprising vector DNA. Another preferred object of the invention is to improve transformation efficiency of a host cell having an R-M system with unmodified transforming DNA comprising a polynucleotide selected from the group comprising plasmids, suicide plasmids, shuttle vectors, cosmids, fosmids, replicons, amplicons, BACs, YACs, and episomes of all types, or mixtures thereof.

Another preferred object of the invention is to improve transformation efficiency of a host cell having an R-M system with unmodified transforming DNA comprising a transposon or an artificial transposon. Still another preferred object of the invention is to improve transformation efficiency of a host cell having an R-M system with unmodified transforming DNA comprising a synaptic complex between an artificial transposon and a transposase, which synaptic complex, upon introduction into the host cell, is capable of catalyzing in vivo transposition of the transposon into DNA present in the host cell.

It is an object of the invention to improve transformation efficiency of a host cell having an R-M system with unmodified transforming DNA comprising a DNA polynucleotide, at least a portion of which has homology to DNA present in the host cell. A preferred object of the invention is to improve transformation efficiency of a host cell having an R-M system with unmodified transforming DNA comprising a PCR amplification product or a synthetic oligonucleotide, at least a portion of which has homology to DNA in the host cell. Another preferred object of the invention is to improve transformation efficiency of a host cell having an R-M system with unmodified transforming DNA wherein the unmodified transforming DNA, upon introduction into the host cell, undergoes homologous recombination with DNA present in the host cell.

It is another object of the invention to obtain transformed cells which result from use of the methods for improving transformation efficiency of the host cells. By way of example, but not of limitation, one object of the invention is to obtain transformed cells having new heritable genotypes and new properties which have commercial applications. Still another object is to obtain transformed cells that contain genes which are capable of expressing proteins that are useful for some commercial purpose using methods of the invention which permit improved transformation of a host cell. One object of this aspect of the invention is to improve transformation efficiency with unmodified transforming DNA of a host cell comprising a lactic acid bacterium having an R-M system.

Another object of the invention is to provide a kit for improving transformation efficiency with unmodified transforming DNA of host cells having an R-M system. A preferred object of the invention is to provide kits for improving transformation efficiency, wherein the kits comprise an inhibitor of the restriction activity of an R-M system of the host cells. A preferred object of the invention is to provide a kit for improving transformation efficiency of a host cell having an R-M system with unmodified transforming DNA, wherein the kit comprises a preparation of an inhibitor which binds to the host cell's R-M system so as to reduce or block restriction of unmodified transforming DNA during transformation and which kit is for use with transforming DNA that is introduced into the host cell by electroporation.

Still other objects of the invention are to provide increased transformation efficiencies of DNA comprising plasmids, fosmids, cosmids, BACs and transposons in bacterial strains having an R-M system selected from among a Type I R-M system, a Type II R-M system, and a Type III R-M system.

It is another object of the invention to provide a protein that inhibits the R-M system of the host cells in vivo during genetic transformation.

Yet another object of the invention is to provide conditions in which unmodified foreign nucleic acids are protected from restriction damage in the host cells during genetic transformation.

Another object of the invention is to provide optimized systems for introduction of active undamaged transposon synaptic complexes comprising unmodified artificial transposons into host cells in order to obtain higher transposition efficiencies in vivo.

Yet another object of the invention is to provide conditions in which unmodified foreign nucleic acids are protected from restriction damage following introduction into host cells in order to obtain transient gene expression of genes on the unmodified foreign DNA.

Yet another object of the invention is to provide a kit for obtaining improved transient expression of genes on unmodified foreign DNA which is introduced into host cells by electroporation, wherein the kit comprises a preparation of an inhibitor of the restriction activity of an R-M system present in the host cells.

A different preferred object of the invention is to provide a method for genetically modifying a cell having an R-M system in order to obtain a host cell having a heritable genotype which encodes a phenotype which permits improved transformation efficiency by unmodified transforming DNA.

One object of the invention is to obtain host cells having a heritable genotype which encodes a phenotype that permits improved transformation efficiency by unmodified transforming DNA for host cells selected from the group consisting of Eubacteria or Archaebacteria. A preferred object is to obtain host cells which have which have a heritable genotype which encodes a phenotype that permits improved transformation efficiency by unmodified transforming DNA and which can be used for commercial applications. Without limiting the invention, one example of this object of the invention is to obtain lactic acid-producing bacteria which have improved transformation efficiency by unmodified transforming DNA.

Another object of this aspect method of the invention is to obtain host cells having a heritable genotype which encodes a phenotype comprising improved transformation efficiency by unmodified transforming DNA for host cells having at least one restriction and modification (R-M) system selected from among a Type I R-M system, a Type II R-M system, or a Type III R-M system.

Another object is to obtain host cells having a heritable genotype which encodes a phenotype comprising improved transformation efficiency by unmodified transforming DNA which is introduced into the host cells by electroporation.

Another object is to obtain host cells having a heritable genotype which encodes a phenotype comprising improved transformation efficiency by unmodified transforming DNA comprising a transposon or an artificial transposon. A preferred object of this aspect of the invention is to obtain host cells having a heritable genotype which encodes a phenotype comprising improved transformation efficiency by unmodified transforming DNA comprising a transposon or an artificial transposon that comprises a synaptic complex between the transposon and a transposase.

Another object is to obtain host cells having a heritable genotype which encodes a phenotype comprising improved transformation efficiency by unmodified transforming DNA comprising a polynucleotide selected from the group comprising plasmids, suicide plasmids, shuttle vectors, cosmids, fosmids, oligonucleotides, replicons, amplicons, BACs, YACs, episomes of all types, or mixtures thereof.

Another object is to obtain host cells having a heritable genotype which encodes a phenotype comprising improved transformation efficiency by unmodified transforming DNA comprising a polynucleotide, at least a portion of which has homology to DNA present in the host cell.

Yet another preferred object of the invention is to provide kits for genetically modifying cells having an R-M system in order to obtain a host cells having a heritable genotype which encodes a phenotype that permits improved transformation efficiency by unmodified transforming DNA.

DETAILED DESCRIPTION OF THE INVENTION

A preferred embodiment of the invention is a method for improving transformation efficiency of host cells having a restriction and modification (R-M) system with an unmodified transforming DNA, said method comprising introducing into the host cell an inhibitor of the restriction activity of the host cell's R-M system so as to reduce or block restriction of the unmodified transforming DNA during transformation.

Host cells of the Invention

Preferred host cells of the invention are host cells selected from among Eubacteria and Archaebacteria. Any host cells having an R-M system which has restriction activity that reduces transformation efficiency of unmodified transforming DNA and for which an inhibitor of said restriction activity can be obtained and introduced into the host cells in active inhibitory form are suitable for use in the methods of the invention. The invention comprises both methods for using any host cells having a prokaryotic R-M system, whether this R-M system is naturally occurring or has been introduced using molecular genetic techniques known in the art, and methods for using eukaryotic R-M-type systems which result in restriction of transforming DNA in the host cell.

As discussed by Noreen E. Murray [Microbiol. Molec. Biol. Rev., 64: 412 434, 2000; Microbiology, 148: 3 20, 2002] and Robert Yuan [Ann. Rev. Biochem., 50: 285 315, 1981], and in the 3 .sup.rd edition of Sambrook and Russell's Molecular Cloning--A Laboratory Manual, all of which are incorporated herein by reference, three types of R-M systems (designated Types I, II and III) are currently known for prokaryotic cells. The present invention comprises methods to improve transformation efficiency by unmodified transforming DNA of Eubacteria and Archaebacteria host cells having at least one R-M system selected from among any of these three types of R-M systems, wherein the transforming DNA has at least one site which, in the absence of an inhibitor for the restriction activity of the host cell, would be restricted in the host cell. Murray [Ibid] and Yuan [ibid] disclose the characteristics and methods (and references therefor) for detecting the presence of and assaying for different types of R-M systems and restriction activities, which methods are incorporated herein by reference. Many of these methods are somewhat laborious and can be complicated, and knowledge of the characteristics and identity of the R-M system(s) in a particular host cell strain may not be a primary interest of an investigator who wishes to transform the host cells for a particular purpose.

The methods of the present invention can be used without the investigator knowing the identity of the R-M system(s) in the host cells used. In fact, there are several reasons why it would be preferable to apply the methods of the present invention without identifying or characterizing or assaying for the R-M system or restriction activity of the host cell used, including: (1) the R-M systems of many strains of prokaryotic host cells are unknown; (2) the R-M systems can vary even for different isolates of the same species of host cell; (3) some R-M systems are encoded by one or more plasmids, which may be transmissible between different strains; and (4) some host cells, such as the previously discussed example of H. pylori, can have more than one R-M system in the same cell.

Fortunately, some inhibitors of restriction activity can be used in methods of the invention for inhibiting the restriction activity of different R-M enzymes in different organisms. For example, some inhibitors can be used to inhibit restriction activity of most or all Type I systems. Thus, in many cases, it is easier to simply use the methods of the invention than to try to identify or characterize the R-M system(s) of the host cells. Further, since, for example, about half of the bacterial genomes examined had genes which encoded polypeptides homologous to Type I R-M systems [Murray, N. E., op cit], indicating that such Type I R-M systems are widespread in nature, there is a high likelihood that use of a method of the invention will be successful in increasing transformation efficiency by unmodified transforming DNA even without knowing the identity or characteristics of the R-M system of the host cells. If the inhibitor used is not effective in inhibiting the restriction activity of the particular host cells used, little additional time will be lost in testing another potential inhibitor of the invention, and overall, little time will be lost even if none of the inhibitors tested is effective. In that case, the investigator can simply try other suitable inhibitors of the invention, such as, but not limited to, the inhibitors described below, in order to inhibit the restriction activity of a particular host cell.

Inhibitors of the Restriction Activity of R-M Systems of the Invention

The present invention comprises any inhibitor of the restriction activity of an R-M system, which inhibitor can be introduced into a host cell so as to improve transformation efficiency of the host cell by unmodified transforming DNA by reducing or blocking restriction of the unmodified transforming DNA during transformation. By way of example, but not of limitation, an inhibitor of the invention can be a negatively charged polypeptide that binds to the host cell R-M system so as to improve transformation efficiency of the host cell by unmodified transforming DNA by reducing or blocking restriction of the unmodified transforming DNA during transformation. A "polypeptide" as used herein refers to a polymer of amino acids; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.

In another embodiment of the invention, the inhibitor can be a polypeptide encoded by a bacteriophage gene that binds to the host cell R-M system so as to improve transformation efficiency of the host cell by unmodified transforming DNA by reducing or blocking restriction of the unmodified transforming DNA during transformation. In a preferred embodiment of the invention, the inhibitor is a polypeptide encoded by a T7-like bacteriophage gene. The genetic organization of all T7-like phages that have been examined has been found to be essentially the same as that of T7. Examples of T7-like phages according to the invention include, but are not limited to Escherichia coli phages T3, phi.I, phi.II, W31, H, Y, A1, 122, cro, C21, C22, and C23; Pseudomonas putida phage gh-1; Salmonella typhimurium phage SP6; Serratia marcescens phages IV; Citrobacter phage ViIII; and Klebsiella phage No. 11 [Hausmann, Current Topics in Microbiology and Immunology, 75: 77 109, 1976; Korsten, et al., J. Gen. Virol., 43: 57 73, 1975; Dunn, et al., Nature New Biology, 230: 94 96,1971; Towle, et al., J. Biol. Chem., 250:1723 1733, 1975; Butler and Chamberlin, J. Biol. Chem., 257: 5772 5778, 1982]. In a most preferred embodiment of the invention, the inhibitor is an OCR protein encoded by the 0.3 ocr gene of bacteriophage T7 or bacteriophage T3. However, the invention is not limited to inhibitor of restriction encoded by T7-like phages and inhibitors encoded by other phages are also intended to be within the scope of the invention. As defined herein, an OCR protein comprises any naturally-occurring polypeptide which is encoded by a bacteriophage genome, wherein said polypeptide overcomes classical restriction of unmodified transforming DNA in a bacterial cell that can be infected by said bacteriophage by binding to an R-M system of said bacterial cell so as to inhibit the restriction activity of said R-M system. Whenever the inventors refer to T7 or T3 OCR Protein, or to the genes therefor, in the specification of the invention herein, it will be understood that the invention also includes other OCR proteins, and their respective genes, wherein said OCR proteins inhibit the restriction activity of an R-M system in a host cell.

In another embodiment of the invention, the inhibitor of the invention can be a negatively charged polypeptide encoded by a naturally-occurring transmissible or conjugative plasmid, which polypeptide binds to the host cell R-M system so as to improve transformation efficiency of the host cell by unmodified transforming DNA by reducing or blocking restriction of the unmodified transforming DNA during transformation. Preferably, the polypeptide is a negatively charged protein which mimics DNA and binds to the R-M enzyme system so as to inhibit restriction activity of said enzyme system in the host cell. In a preferred embodiment, the inhibitor is a polypeptide encoded by a gene selected from among an ardA gene, an ardB gene, and an ardC gene [Belogurov, A. A. et al., J. Mol. Biol, 296: 969 977, 2000]. Belogurov et al. reported that several ArdA, ArdB, and ArdC anti-restrictive proteins have a common "anti-restriction" domain, which appeared to correlate with anti-restriction activity of the proteins [ibid; Belogurov, A. A., and Delver, E. P., Nucleic Acids Res., 23: 785 787, 1995]. As defined herein, an Ard protein comprises any polypeptide which is encoded by a naturally-occurring transmissible or conjugative plasmid or replicon, wherein said polypeptide, when present in a Eubacterial or Archaebacterial host cell, alleviates, reduces or blocks restriction of unmodified DNA which, except for the presence of said polypeptide, would be restricted by the restriction activity of an R-M system of said host cell. Whenever the inventors refer to ArdA, ArdB, or ArdC proteins, or to the genes therefor, in the specification of the invention herein, it will be understood that the invention also includes other Ard proteins, and their respective genes, wherein said Ard proteins inhibit the restriction activity of an R-M system in a host cell. An inhibitor of the invention can comprise any acidic or negatively-charged polypeptide, wherein said polypeptide comprises at least one amino acid domain having an amino acid sequence and three-dimensional structure, wherein said domain binds to the R-M system so as to inhibit its restriction activity with respect to unmodified transforming DNA. Preferably, the inhibitor is added to competent host cells prior to or concurrent with addition of the transforming DNA. Most preferably, the inhibitor is added to electrocompetent host cells and both the inhibitor and the transforming DNA are incorporated into the host cells by electroporation.

It has been reported that both the T7 0.3 ocr gene product and ArdA protein inhibit multiple Type I R-M enzymes [In: Murray, N. E., Microbiology, 148: 3 20, 2002]. However, some embodiments of the invention, especially when the R-M system of the host cell is unknown and/or in cases where there are known to be or may be more than one R-M system in a particular host cell, more than one inhibitor of restriction activity of the R-M systems of the host cell are used simultaneously to improve transformation efficiency by unmodified transforming DNA of the invention.

Another embodiment of the invention is an inhibitor of restriction comprising a protein which is responsible for restriction alleviation in a cell, as discussed by Noreen Murray [Microbiol. Molec. Biol. Rev., 64: 412 434, 2000 (especially pp. 423 425)], incorporated herein by reference. Thus, one embodiment of the invention is an inhibitor comprising a protein responsible for alleviation of restriction activity wherein the protein is ClpXP protease encoded by clpX and clpP genes.

Although the inhibitors described herein above are preferred inhibitors of restriction activity of an R-M system of the invention, the invention is not limited to these inhibitor polypeptides. The inhibitor can be a polypeptide encoded by any gene in a naturally-occurring bacteriophage or in an autonomously-replicating DNA molecule, such as a transmissible or conjugative plasmid, in a Eubacterium or an Archaebacterium, wherein said inhibitor improves transformation efficiency of the host cell by unmodified transforming DNA by reducing or blocking restriction of the unmodified transforming DNA during transformation.

Inhibitors of the present invention are not limited to polypeptide molecules, although negatively charged polypeptide molecules are preferred inhibitors of the invention. Thus, an inhibitor of the invention can also be a nucleic acid, polynucleotide, oligonucleotide or a segment of a nucleic acid or polynucleotide, including nucleic acids composed of either DNA or RNA, or both DNA and RNA mononucleosides, including non-naturally occurring DNA or RNA mononucleosides. [The inventors use the term "non-naturally occurring" herein to refer to nucleic acid bases, sugars and internucleoside linkages which are not those which are common in most cells in nature, even though some of them could occur naturally in some cells or under some conditions; this term is used to minimize the use of the term "modified" with respect to such bases, sugars or internucleoside linkages in order to avoid confusion with "unmodified" transforming DNA of the invention. As discussed earlier, the modification enzymes of an R-M system that protect the DNA from digestion are generally DNA methyltransferases which catalyze methylation of specific nucleic acid bases within the target sequence that is digested by the endonuclease, resulting in DNA that is "modified" with respect to that R-M system. The term "unmodified transforming DNA" as used herein refers to DNA that lacks methyl groups or other modifications, which usually, but not always, have been added by the modification activity of an R-M system.]

The invention does not limit the composition of the nucleic acids or polynucleotides comprising inhibitors of the restriction activity of an R-M system so long as each said nucleic acid functions for its intended use. Preferably the oligonucleotide or polynucleotide improves transformation efficiency of the host cell by unmodified transforming DNA by reducing or blocking restriction of the unmodified transforming DNA during transformation. For a variety of reasons, a nucleic acid or polynucleotide of the invention may comprise naturally-occurring nucleic acid bases, sugar moieties, or internucleoside linkages or one or more non-naturally occurring nucleic acid bases, sugar moieties, or internucleoside linkages. By way of example, one reason for using nucleic acids or polynucleotides that contain non-naturally occurring bases, sugar moieties, or internucleoside linkages is to change the susceptibility of the polynucleotide to one or more nucleases. By way of example, the non-naturally occurring portions of the polynucleotide or oligonucleotide can include, but are not limited to, one or more phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphordiamidate, methylphosphonate, alkyl phosphotriester, phosphoroselenate, phosphorodiselenate or formacetal, or analogs thereof, inter-sugar (backbone) linkages, some of which are resistant to some nucleases.

With respect to nucleic acids or polynucleotides of the invention, one or more of the sugar moieties can comprise ribose or 2'-deoxyribose, or alternatively, one or more of the sugar moieties can be some other sugar moiety, such as, but not limited to, 2'-fluoro-2'-deoxyribose or 2'-O-methyl-ribose, which provide resistance to some nucleases.

The invention also does not limit the composition of the nucleic acid bases in polynucleotides or oligonucleotides comprising inhibitors of the restriction activity of an R-M system so long as each said nucleic acid functions for its intended use. By way of example, but not of limitation, the nucleic acid bases in the mononucleotides may comprise guanine, adenine, uracil, thymine, or cytidine, or alternatively, one or more of the nucleic acid bases may comprise xanthine, allyamino-uracil, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl adenines, 2-propyl and other alkyl adenines, 5-halouracil, 5-halo cytosine, 5-propynyl uracil, 5-propynyl cytosine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-methyl-adenine, 7-deaza-7-methyl-guanine, 7-deaza-7-propynyl-adenine, 7-deaza-7-propynyl-guanine and other 7-deaza-7-alkyl or 7-aryl purines, N2-alkyl-guanine, N2-alkyl-2-amino-adenine, purine 6-aza uracil, 6-aza cytosine and 6-aza thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo adenine, 8-amino-adenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines and 8-halo guanines, 8-amino-guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8 substituted guanines, other aza and deaza uracils, other aza and deaza thymidines, other aza and deaza cytosine, aza and deaza adenines, aza and deaza guanines or 5-trifluoromethyl uracil and 5-trifluorocytosine. Still further, they may comprise a nucleic acid base that is derivatized with a biotin moiety, a digoxigenin moiety, a fluorescent or chemiluminescent moiety, a quenching moiety or some other moiety. The invention is not limited to the nucleic acid bases listed; this list is given to show the broad range of bases which may be used for a particular purpose in an assay.

A variety of methods are known in the art for making nucleic acids having a particular sequence or that contain particular nucleic acid bases, sugars, internucleoside linkages, chemical moieties, and other compositions and characteristics. Any one or any combination of these methods can be used to make a nucleic acid, polynucleotide, or oligonucleotide for the present invention. Said methods include, but are not limited to: (1) chemical synthesis (usually, but not always, using a nucleic acid synthesizer instrument); (2) post-synthesis chemical modification or derivatization; (3) cloning of a naturally occurring or synthetic nucleic acid in a nucleic acid cloning vector (e.g., see Sambrook, et al., Molecular Cloning: A Laboratory Approach 2.sup.nd ed., Cold Spring Harbor Laboratory Press, 1989) such as, but not limited to a plasmid, bacteriophage (e.g., m13 or lamda), phagemid, cosmid, fosmid, YAC, or BAC cloning vector, including vectors for producing single-stranded DNA; (4) primer extension using an enzyme with DNA template-dependent DNA polymerase activity, such as, but not limited to, Klenow, T4, T7, rBst, Taq, Tfl, or Tth DNA polymerases, including mutated, truncated (e.g., exo-minus), or chemically-modified forms of such enzymes; (5) PCR (e.g., see Dieffenbach, C. W., and Dveksler, eds., PCR Primer: A Laboratory Manual, 1995, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); (6) reverse transcription (including both isothermal synthesis and RT-PCR) using an enzyme with reverse transcriptase activity, such as, but not limited to, reverse transcriptases derived from avian myeloblasosis virus (AMV), Maloney murine leukemia virus (MMLV), Bacillus stearothermophilus (rBst), Thermus thermophilus (Tth); (7) in vitro transcription using an enzyme with RNA polymerase activity, such as, but not limited to, SP6, T3, or T7 RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, or another enzyme; (8) use of restriction enzymes and/or modifying enzymes, including, but not limited to exo- or endonucleases, kinases, ligases, phosphatases, methylases, glycosylases, terminal transferases, including kits containing such modifying enzymes and other reagents for making particular modifications in nucleic acids; (9) use of polynucleotide phosphorylases to make new randomized nucleic acids; (10) other compositions, such as, but not limited to, a ribozyme ligase to join RNA molecules; and/or (11) any combination of any of the above or other techniques known in the art. Oligonucleotides and polynucleotides, including chimeric (i.e., composite) molecules and oligonucleotides with non-naturally-occurring bases, sugars, and internucleoside linkages are commercially available (e.g., see the 2000 Product and Service Catalog, TriLink Biotechnologies, San Diego, Calif., USA; www.trilinkbiotech.com).

There are a variety of methods for finding an oligonucleotide or polynucleotide which has activity as an inhibitor of the restriction activity of an R-M system according to the invention. For example, since the region of OCR Protein that binds to the restriction domain of a Type I R-M system has been shown to resemble the bent structure of DNA bound to the Type I R-M complex [Atanasiu, O. B., et al., Nucl. Acids Res., 29: 3059 3068, 2001; Walkinshaw, M. D., et al., "Structure of ocr from bacteriophage T7, a protein that mimics B-form DNA," Molec. Cell, 9: 187 194, 2002] and also, because the nucleotide recognition sequences for the restriction activity of some R-M systems is known, an inhibitor of restriction activity of the R-M system can be made by rational design in some cases. By way of example, an oligonucleotide can be made which has similar structure and nucleic acid base sequence to that which is recognized by the active site of the restriction enzyme domain of the R-M system, but wherein the oligonucleotide comprises modified sugar moieties and/or internucleoside linkages, as discussed herein, which cannot be digested by the restriction enzyme.

Alternatively, a method termed "SELEX," as described by Gold and Tuerk in U.S. Pat. No. 5,270,163, can be used to select a nucleic acid for use as an inhibitor according to the invention. SELEX permits selection of a nucleic acid molecule that has high affinity for a specific analyte from a large population nucleic acid molecules, at least a portion of which have a randomized sequence. For example, a population of all possible randomized 25-mer oligonucleotides (i.e., having each of four possible nucleic acid bases at every position) will contain 4.sup.25 (or 10.sup.15) different nucleic acid molecules, each of which has a different three-dimensional structure and different analyte binding properties. SELEX can be used, according to the methods described in U.S. Pat. Nos. 5,270,163; 5,567,588; 5,580,737; 5,587,468; 5,683,867; 5,696,249; 5,723,594; 5,773,598; 5,817,785; 5,861,254; 5,958,691; 5,998,142; 6,001,577; 6,013,443; 6,030,776; and 6,300,074, incorporated herein by reference, in order to select an analyte-binding nucleic acid with high affinity for the restriction activity domain of the R-M system of the host cell. A polynucleotide or oligonucleotide inhibitor of the invention that is obtained using SELEX may comprise naturally occurring nucleic acid bases, sugar moieties, or internucleoside linkages or one or more non-naturally occurring nucleic acid bases, sugar moieties, or internucleoside linkages.

By way of example, but not of limitation, SELEX can be used to find an inhibitor of restriction activity as follows: If the R-M system and a suitable nucleic acid substrate for which the R-M system has restriction activity can be obtained in purified form, then the substrate can be incubated with an active form of the R-M system under suitable restriction activity reaction conditions and the restriction activity of the R-M system on the substrate can be assayed in vitro by gel electrophoresis. Then, for example, all possible randomized 25-mer oligonucleotides can be screened using the SELEX procedure to find suitable candidate oligonucleotide inhibitors that inhibit the restriction activity of the R-M system. Among those candidates, the best in vitro inhibitors can be screened by in vivo transformation in order to find inhibitors that increase transformation efficiency of a host cell by unmodified transforming DNA having a selectable marker, such as an antibiotic resistance marker. By way of example, but not of limitation, an inhibitor of restriction activity of an R-M system of the invention that comprises DNA and/or RNA can be obtained using SELEX in a manner similar to that described for obtaining an inhibitor for Taq and Tth DNA polymerases [U.S. Pat. No. 6,020,130]; these inhibitors of Taq and Tth DNA polymerase activity exemplify that SELEX can be used to obtain an inhibitor for an enzyme that interacts with a DNA molecule, which is also the case for an inhibitor of restriction activity of an R-M system of the present invention. Further, those with skill in the art will know that DNA, including DNA that is an inhibitor of the restriction activity of an R-M system that is selected using SELEX according to the invention, can be used to transform a chemically-competent host cell, or to transform an electrocompetent host cell by electroporation, or to transform other cells by other means, using methods similar to those known in the art.

Once selected using SELEX, nucleic acid inhibitor molecules can be made by any of numerous known in vivo or in vitro techniques, including, by way of example, but not of limitation, automated nucleic acid synthesis techniques, PCR, or in vitro transcription, including in vitro transcription using T7 R&DNA.sup.TM. Polymerase (EPICENTRE), which incorporates non-canonical nucleotides as well as canonical nucleotides [U.S. Pat. Nos. 5,849,546; and 6,107,037].

In still another embodiment of the invention, the inhibitor of restriction activity that improves transformation efficiency o