Purification of a triple heli formation with an immobilized oligonucleotide

Description

Method for purifying triple hexadecimal formations in a cyclic manner The invention relates to a novel method for purifying DNA. The method of the invention enables the rapid purification of pharmaceutically usable double-stranded DNA. More specifically, the method of the invention involves specific hybridization between a DNA sequence and an oligonucleotide. Gene and cell therapy techniques: these days, they are undergoing remarkable development. These techniques have the potential to produce large quantities of pharmaceutical-grade DNA. In these new therapies, the drug often contains the DNA itself, and it is essential to produce, isolate and purify it in sufficient quantities to be used therapeutically in the human body. In recent years, several reports have demonstrated the feasibility of injecting plasmid DNA for therapeutic or vaccination purposes, demonstrating that various cell types are able to take up DNA expression vectors and subsequently express genes encoded by these plasmids [Leöley, ffom. Gene Ther. 6,1129 (1995)]. Genes used in gene therapy or vaccination may include, for example, tumor suppressor genes, suicide genes, or antisense sequences. They may also encode proteins such as alpha-fetoprotein (AFF) [Morinaga, Proc. Natl. Acad. Sci. USA 80, 46Θ4 (1983)], enzymes, hormones, chokines, growth factors such as FGF [Jouannean et al., Proc. Natl. Acad. Seb USA 88, 2893 (1991)] or YEGFS (Olofsson et al., Proceedings 93, 576 (1996)], or wild-type factors such as B~delete factor VIII (Ttueít et sl., DNA 4, 333 (1985)], apolipoproteins, neurotransmitters, neurotrophic factors, natural or artificial immunoglobulin. Reporter genes, such as laeZ encoding fiericfá& coő 3-galactosidase, are also useful. The main challenges in the use of plasmid DNA, a vector carrying a gene, in the human body are the (i) production and (ii) purity of this product. The production of plasmid vectors in large numbers in the Awfevfeh / o coöf host has recently been achieved. The plasmids or Co!Et-derived plasmids used today are pBR322, pUC or pBlttescript [Lahijam et al, Hum. Gene Ther. .7, 197! (199(5) and W1NNACKER EE: TRGM GENES TO CLONES'TROM GENES TG CLONES. "INTRODUCTION TG GENE TECHNOLOGY", ed.: WFJNHEIM, VC1-I VERÍAGSÖESELESCHAFRDE, 1987, pp. 125-132.] or pCOR plasmids [Souhrier et &!., Gene Therapy 6, 1482 (1999)]. The second concern with the use of plasmid DNA as a gene therapy vector is the purity of the plasmid vector. Currently used purification methods, such as ultracentrifugation with a CsCl gradient or chromatography, are not always effective in removing contaminants such as host genomic DNA and RNA or proteins. It is particularly difficult to remove host genomic DNA using classical chromatography because its chemical structure is very close to that of plasmid DNA. Plasmid preparations obtained by classical chromatography typically contain 0.5-1% host genomic DNA. Therefore, in order to develop plasmid DNA as a safe vector for gene therapy, purification technologies are needed that reduce the host genomic DNA content to much lower levels, typically 0.1%, or even 0.01% or even lower. The invention relates to a simple and particularly efficient new DNA purification process. This enables particularly high purity to be achieved with high yield. The process according to the invention is essentially a purification process. 108320-2709 ET / An ο DNA is synthesized by specific interactions between a sequence and an oligonucleotide consisting of natural or modifier bases. It has been shown that certain oligonucleotides can interact specifically in the groove of the DNA double helix, locally forming a triple helix, which leads to the inhibition of transcription of genes [Héléae et Tónimé, Biocheim. Biophys. Acta 99, 1049 (1990)]. These oligonucleotides selectively recognize the double helix as oligopurine-oligoperamide sequences—in regions where one strand contains an oligopurine sequence and the complementary strand contains an oligopurine sequence—and locally form a triple helix. The bases of the third strand (the oligonucleotide) form hydrogen bonds (Hoogsteen or reverse Hoogsteen bonds) with the Watson-Crick-like base pairs. The use of this type of interaction for plasmid isolation has been previously described. WO90 18744 (Rhone Poulene Korer SA) discloses the isolation of plasmids by triple helix hybridization with oligonucleotides directed against the replication origin. WO994907 (Rhone Poulene Korer S.A.) discloses special oligonucleotides that bind to plasmid sequences by triple helix hybridization. For example, L'to et al. (PNAS 89,495 (1992)) have reported the use of a monoclonal antibody capable of recognizing certain plasmid sequences and the formation of a triple helix with it. The complexes thus formed are then brought into contact with magnetic beads coated with streptavidin. The interaction between the monoclonal antibody and streptavidin then allows the isolation of the plasmid by magnetic separation following the precursor of the beads. However, this method has some disadvantages.Two sequential specific interactions are required, the first between the oligonucleotide and the plasmid, and the second between the biotinylated complex and the streptavidin beads. Furthermore, the final solution may be contaminated with biotinylated oligonucleotides and thus cannot be used as a pharmaceutical preparation. The invention relates to a new, improved DNA purification process which utilizes this type of interaction. More specifically, the process according to the invention utilizes oligonucleoside covalently linked to a carrier. This process is particularly rapid, results in high yields and a high degree of purity. It also allows the purification of DNA from complex mixtures containing other nucleic acids, proteins, eodotoxins (such as lipopolysaccharides), enzymes and the like. The carriers used are also easily renewable and the DNAs obtained exhibit improved drug safety properties. Finally, this process, in contrast to previous methods, consists of only one step. Therefore, a first embodiment of the invention is a double-stranded DNA purification method in which a solution containing said DNA mixed with other components is passed through a column to which an oligonucleotide forming a triple helix by hybridization with a specific sequence of said DNA is covalently linked. The specific sequence may occur naturally in double-stranded DNA or may be an artificially introduced synthetic sequence. The nucleotides used in the invention are oligonucleotides that directly hybridize to double-stranded DNA. These oligonucleotides may contain the following bases: thymine (T), which can form a triplet with the AT doublet of double-stranded DNA {Rajagopai et al., Biochem. 2g, 7859 (1989)}; » adenine (A), which can form a triplet with the AT doublet of double-stranded DNA; • guanine (g), which can form a triplet with the GC doublet of double-stranded DNA; • protonated leucine (Ca), which can form a triplet with the GC doublet of double-stranded DNA (Rajsgopal et ah. Biochem. 28, 7859 (1989)]; * their guards (G), which can form a triple with AG or AT base pairs. The oligonucleotide used preferably contains a cytosine-rich homopyrimidine sequence and the specific sequence present in DNA is the homopurine-homopyrimidine sequence. The presence of cytosines allows the formation of a triple helix, which is stable at acidic pH, where cytosines are in a protonated state, and destabilized under alkaline conditions, where cytosines are neutral. In order to be able to create a triple helix by hybridization, it is important that the oligomer and the specific sequence marked in the DHS are complementary to each other. In this context, in order to achieve the best yield and the best selectivity, the method according to the invention uses an oligomer and a specific sequence that are completely complementary to each other. This can be mainly: poly(CTT) oligomer and poly(GAA) special sequence. As an example, the following sequences can be mentioned: 5--GAGGCTTCTT€TrCTTCTTCTTC'fT«3XGAGG(CTT)7;. SEQ ID NO: 1), in which the GAGG bases do not form a triple helix but allow the oligonucleotide to be assembled apart from the linker; (CTT)7(SEQ ID NO: 26) may also be mentioned. These oligonucleotides are capable of forming a triple helix with a specific sequence containing complementary units (GAA). The sequence in question may be mainly a region containing 7, 14 or 1 7 GAA units, as described in the examples. Another particularly important sequence is SAAGGAGGAGGAGGAGAGGA-S' (SEQ ID NO: 5). This sequence forms a triple helix with the nucleotides SAAGGAGGAGGGAGGGAGAG-3' (SEQ ID NO: 6) or 5'-TTGGTGTGGTGGGTGGGTT-3' (SEQ ID NO: 7). In this case, the oligonucleotide binds to the polypurine strand in an antiparallel orientation. These triplexes are stable only in the presence of Mg [Vasguez et al., Biochem. 34, 7243-7251 (1995); Seal and Dervass, Science 251, 1360-1363 (1991)], As mentioned above, the specific sequence may be a sequence naturally occurring in double-stranded DNA or a synthetic sequence introduced later. It is particularly advantageous to use an oligonucleotide which is capable of forming a triple helix with a sequence naturally occurring in double-stranded DNA, for example in the replication origin or marker gene of a plasmid. In this context, plasmid sequence analysis has shown that some regions of these DNAs, particularly in the replication origin, have homopurine-homopyrimidine regions. The synthesis of oligonucleotides capable of forming a triple helix with these natural homopurine-homopyrimidine regions advantageously allows the application of the method according to the invention to unmodified plasmids, such as commercially available pUC, pBK322, pSV and the like. Among the homomeric-comomeric sequences occurring naturally in double-stranded DNA, ColEl .£ can be mentioned.ofo' a sequence present in the origin of replication of a plasmid, containing in whole or in part the sequence 5'-€TTCCCGAAGGGAGAAAGG-3' (SEQ ID NO: 2). In this case, the oligo forming the triple helix has the base sequence: 5GAAGGGCTTCCCTCTTTCG-3' (SEQ ID NO: 3) and is alternately linked to the core strand of the double helix as described by Seal and Derya® p, Arn. Cher®, Soc. 114, 4976-4982 (1992)] and Jayassna and .fohnsion [Noel. Acids Res, 20, 5279-5288 (1992)). The sequence 5'-GA,AfkAAGGAAGAG-3' of the β-lactisase gene of plasmid pBR322 may also be mentioned (Dw^Yatafe et al., Ents. Nstl Acad. Sci. USA 89, 504-508 (1992)). Two additional primer sequences were identified in the ColEl and pCOR replication origins, which are capable of forming a triplex structure with some oligonucleoside residues. Plasmids derived from ColEl contain a 12-phage homoporin sequence (S'-AGAAAAAAAGGA-o') (SEQ ID NO: 27) upstream of the RNA11 transcript in plasmid replication (Lscatena et al., Nature 294, 623 (1935)). This sequence forms a stable triplicate structure with the 12-membered complementary oligo S-TCTTf'TTTTCCT-S' (SEQ ID NO: 23). The pOOR framework contains a 14-base homoporin repeat (5'-AAGAAAAAAAA.<3AA-3*} (SEQ ID NO: 29), which is located in the A + T-rich segment of the pCOR. y origin of replication [Leveheo.ko et al., Noel. Aeíds Res. ;24. 1936 (1996)], This sequence forms a stable triplex structure with the i4-membered complementary oligonucleotide 5'-TTCTTTTTTTTCTT-3'' (SEQ ID NO: 30), The corresponding oligonucleotides. 5TCTTTTTTTCCT-3'' (2S.(SEQ ID NO: 39) and 5'-TTΟΟΓΤΤΤ7ΤΕΤΤ-3' (SEQ ID NO: 39) efficiently and specifically target their corresponding complementary sequences located in either the ColEi (guard) or pCÖR (replication) origins. In fact, a single o«tn-ka»@Btk»s toid (T*GC or C*AT) can result in complete destabilization of the s iripiet structure. The use of an oligonucleotide capable of forming a triplex with a sequence of interest in an origin of replication or marker gene is particularly advantageous since it allows purification of any DNA containing said origin of replication or said marker gene with the same oligonucleotide. Therefore, it is not absolutely necessary to construct a plasmid or double-stranded DNA for the insertion of an artificial specific sequence. Although fully complementary sequences are preferred, some mismatches between the nucleotide sequence and the sequence present in the DNA can be tolerated, provided that this does not result in a significant loss of affinity. An example is the sequence 5'AAAAAAGGg0AAIAAGOG-3' present in the E. coli β-lactamase gene (SEQ ID NO: 3). In this case, the thymine interrupting the polypurine sequence can be recognized by a guar on the third strand, thereby forming a G*TA triplicate, which is stable when flanked by two T*AT triplets (Ktessling et al., Biol. 34, 2829-2334 (1992)]. In a specific embodiment, the oligonucleotides of the invention comprise the sequences (CCT)n, (CT)n or (CTT)n, where n is a number between 1 and 35. It is particularly advantageous to use sequences of the type (CT)n or (CTTin). It was found that the purification yield was actually influenced by the C content of the oligonucleotide. As shown in Example 7, the purification yield increases when the oligonucleotide contains less C. It should be noted that the oligonucleotides of the invention may also combine (CCT), (CT) or (CTT) units. The antinucleotide used may be natural (unmodified, composed of natural bases) or chemically modified. The antinucleotide may advantageously have certain chemical modifications that allow its resistance to or protection against nucleosides or increase its affinity for a specific sequence, According to the invention, oligonucleotides are understood to mean consecutive, linked nucleosides in which the backbone is modified for resistance to nucleases. Possible modifications include oligonucleotide phosphoramidates, which are capable of forming a triple helix with DNA (Xodo et al., Noel, Adás Res. 22, 3322-3330 (1994)], as well as oligonucleotides with a formatecal or methyl-phosphonate backbone (Matteucd et al., I Am, Chem, Soc, 113, 7767-7768 (1991)]. Nucleotides are derived from the monomer, and oligonucleotides can also be used, which also form a triple helix with DNA (Le Doan et al, Nucl. Aids Res. 15, 7749-7760 (1937)). Another modification of the backbone is the phosphoramidate bond. For example, the one described by Gryaznov and Cheo N3'-P5' nucleotides share a phosphoramidate bond, which allows the nucleotides to form a particularly stable triple helix with BNS [p, Am. Chem. Soc, Uk, 3143-3144 (1994)].Other modifications of the skeleton include ribonucleotides. 2'-O-Medl-rihez, iso-diester, etc. (Sun and Bélése, Cttrr, Opím Simet. Biol. 116, 3143-3144)] can be mentioned. Their end of the phosphorus-based backbone can be replaced by a polymer backbone, such as peptide nucleic acids (PNAs), which can form triple helices in layers [Nieisen et al,, Science 259, 1497-35W (1991); Kim cí ab, J. Am, Ctea. Sec. 115, 6477-0481 (1993)] or a guanidine-based backbone, such as deoxyribonucleic guanidine (DNGs) (Proc. Nafi. Acad. Sri. USA 92, 6097-611 (1995)] or polycyclic DNA with analogues, which also form triple helices. The third strand thymine can also be replaced with H-bromomethyl, which increases the affinity of the oligonucleotide for DNA [Eovsic and Dervan, J. Am. Chem. Soc. 1.1.1, 3059-3061 (1989)]. The third strand may also contain unnatural bases, including ?-dezaza~2'-d«zoxixantozm (Mílligan et ah, Noel Aekís Kés, 21, 327-333 (1993)}, 1-(2~-0£2θχΙ-0-1>ϊΓ!Χϊ^η<κ<ί1>-3-χη«ί11-5-3ίη!ηο-1Α-ρΐ5ί!3θ1[43űjpyrpheidín--7~on (Köb and Bervan, I Am. Chem. Soc. 1.14, 1470-1478 (1992)]), S-oxo-aderun, 2-ammo-ptrine, 2'-O-methyl-pseodolactidia or any other modification known to the skilled person (for a review see San and Bélése, Cárt. Opin. Símet. Bio). 3, 345-356 (1993)]. Other types of modification of the oligonucleotide are aimed in particular at improving the interaction and / or affinity between the oligonucleotide and the specific sequence. The most preferred modification according to the invention consists in the methylation of the oligonucleotide cytosines (see Example 5). The oligonucleotide thus methylated has the remarkable property of forming a stable hex to the specific sequence in near-neutral pH ranges (> 5). This makes it possible to work at higher pH values ​​than those used with previous oligonucleotides, i.e. at pH values ​​where the risk of degradation of plasmid DNAs is much lower. The length of the oligonucleotide used in the method according to the invention is at least 3 bases, preferably 5-30 bases. Oligonucleotides longer than 10 bases are preferably used. The skilled person can determine the length of the oligonucleotide for each case, taking into account the desired selectivity and stability of the interaction. The phenolic compounds of the invention can be synthesized by any known technique. In particular, they can be prepared using trisclemic acid synthases. Obviously, any other method can also be used by the skilled person. The oligonucleotide is generally provided with a functional group for covalent attachment to the support. This can be modified with a chain-end (terminal) thiol, amine or carboxyl group at the 5' or 3' position. In particular, the addition of a mercapto-, iso- or carboxyl group allows the attachment of the oligonucleotide to a support having disulfide, maleimide, amine, carboxyl, ester, epoxy, bromide or aldehyde functional groups. These linkages are formed by the formation of disulfide, thioether, ester, amide or amine bonds between the oligonucleotide and the support. Any other method known to the person skilled in the art can be used, such as, for example, bifunctional coupling reagents. Furthermore, in order to improve hybridization with the linked oligonucleotide, it may be advantageous if the oligomtkieotide contains some "arm" and "gap" base sequence. The use of an arm in fact allows the oligonucleotide to be attached to the support at a selected distance, which improves the conditions for interaction with the DNA. The arm preferably consists of a linear carbon chain containing 1-18, preferably 1-12 (CFT) groups and an amine that allows its attachment to the column. The arm is linked to the oligonkleofid via a phosphate group or to a "gap" consisting of bases that do not interfere with hybridization. Thus, the "gap" may contain porin bases. The "gap" may for example comprise * * « » «· *·» #ίφ ΦΦ* * * * φ ♦ The AGG sequence preferably consists of a linear chain containing 6-12 carbon atoms. Various types of supports can be used to implement the invention. These can be functional chromatographic supports, either bulk or packed into columns, functional chromatographic surfaces or functional latex beads, magnetic or other. Chromatographic supports are preferably used. Chromatographic supports can be, for example, agarose, acrylamide or dextran as well as their derivatives (such as Sephadex, Sepharose, Superose, etc.), polymers such as poly(poly(phenylene)benzene) or grafted or non-grafted silicone. Chromatographic columns can operate in diffusion or perfusion mode. In order to achieve a better purification efficiency, it is particularly advantageous to use several sequences on the plasmid which contain hybridization positions with the oligonucleotide. The presence of several hybridization positions actually promotes interactions between the said sequence and the oligonucleotide, which leads to an improvement in the purification efficiency. Therefore, for an oligonucleotide which contains n (CCT>, (CT) or (C'IT) repeating motifs, it is preferable to use a DNA sequence which contains at least n, and preferably n+1 complementary motifs. A sequence containing n+1 complementary motifs thus provides the oligonucleotide with two hybridization positions. The DNA sequence preferably contains at least 31 hybridization positions, i.e. n-18 complementary motifs. The method of the invention can be used for the purification of any type of double-stranded DNA. An example of the latter is circular DNA, such as a plasmid, which generally carries one or more therapeutically important genes. This plasmid can also contain an origin of replication, a marker gene and the like. The method of the invention can be used directly on cell lysates. In this embodiment, the plasmid multiplied by the secondary culture following transformation is purified directly after lysis of the cells. The method of the invention can also be used on pure lysates, i.e. the supernatant obtained after neutralization and centrifugation following cell lysis. It is obvious that it can also be used on solutions pre-purified by other known methods. This method allows the purification of both linear and circular DNA carrying the sequence in question from a mixture containing different DNAs. The method of the invention can also be used for the purification of double-stranded DNA, The cell lysate may be a lysate of prokaryotic or eukaryotic cells. With regard to prokaryotic cells, E. coli or Streptococcus bacteria may be mentioned as examples. With regard to eukaryotic cells, animal cells, yeasts, fungi and the like are examples, more specifically, K. coli or Streptococcus yeasts or COS, CHO. C127, NIN.3T3 and similar cell lines, The method according to the invention is particularly advantageous because it allows for the rapid and simple recovery of highly purified plasmid DNA. As illustrated in the examples, this method allows for the efficient separation of the plastid DNA in question from contaminating components such as fragmented chromosomal DNA, endotoxins, proteins, nucleases and the like. More specifically, the method according to the invention allows for the recovery of double-stranded DNA, especially of plasmid origin, with a chromosomal DNA content of 8.5% or less. Even more specifically, the recovered DNA has a chromosomal DNA content of 8.2% or less. Therefore, the invention describes compositions containing pharmaceutically suitable plasmid DNA, especially for use in gene therapy or cell therapy. In this context, the invention also relates to a pharmaceutical composition containing linear or plasmid-derived double-stranded DNA prepared according to the following method. Also included in the invention are 8.5% or less, preferably 0.2% or less, ».**« «φ *** «« even more preferably 0.1% or less and even more preferably 0.01% or less of chromosomal DNA are plasmid DNA preparations. According to the following examples: a triplex affinity intermediate step was incorporated into the purification process after classical chromatography steps. This affinity step significantly improves the purity of the plasmid preparation, whatever its initial purity. The formation of a triplex structure between the oligonucleotide (covalently bound to the chromatography column) and the plasmid in question to be purified is based on the fact that the plasmid has a sequence that is capable of forming a triplex structure with this oligonucleotide. This triplex structure is stable only at acidic pH, where the oligonucleotide residues are protonated. The plasmid DNA can then be simply eluted from the column by raising the pH to neutral. The compositions may contain plasmid 5 combined with "cage" or transport carriers—such as liposomes, nanoparticle particles, positively charged lipids, polymers, recombinant viruses or bacteria, and the like. In one embodiment, the method of the invention can be used to purify a specific type of double-stranded DNA from a mixture containing two or more different types of double-stranded DNA and sequences. This method can be directly applied to a cell lysate in which the double-stranded DNA has been amplified by cell culture and purified after lysis of the propagated cells. This method can also be applied to a purified lysate, i.e. the supernatant obtained after neutralization and centrifugation of the cell lysate. The method can also be applied to a pre-purified solution. More specifically, a method for purifying the first double-stranded DNA from a solution containing first and second double-stranded DNA comprises (i) passing the solution through a first column containing a covalently bound oligonucleotide capable of forming a triplex with the second double-stranded DNA by hybridization with a specific sequence contained therein, (ii) recovering the solution passed through the first column enriched in unbound first double-stranded DNA, and (iii) passing the recovered solution through a second column containing a covalently bound oligonucleotide capable of forming a triplex by hybridization with a specific sequence of said first double-stranded DNA. After a suitable washing step, the first double-stranded DNA can be eluted from the second column. By performing this two-step purification procedure, the first double-stranded DNA can be recovered from the second column free of detectable amounts of second double-stranded DNA. In a specific embodiment of the invention, the first double-stranded DNA molecule is the pCOR plasmid having the sequence 5'AAGAAAAAAAAA-3'' (SEQ ID NO: 29), which forms a stable triplex structure with the oligonucleotide having the sequence 5'ITCTTTTI'TTTCTT-3' (SEQ ID NO: 30). The second double-stranded DNA molecule is the CoEl-derived plasmid having the sequence 5'AGAAAAAAAGGA-3' (SEQ ID NO: 27), which forms a triplex with the oligonucleotide having the sequence 5'-TCTTTTTTTCÜT-o' (SEQ ID NO: 28). Accordingly, the pCOR plasmid can be advantageously purified from a solution containing other plasmids, such as a plasmid derived from ColEl, using the two-step purification method described above. The invention will be described in more detail with the help of the following examples, which serve as illustrations and do not represent a limitation of the scope of the invention. General cloning and molecular biology techniques / 1 conventional molecular biology methods, such as restriction enzyme digestion, gel electrophoresis, transformation into L. oofi, preparation of autologous acids and the like, are described in the literature [Maniatis, Fritscfe and Sambrook, Molecular cloning: A laboratory technique, 2nd ed., Cold Spring Harbor Laboratory Press, NY (1989); Ausubei et al., Critical proteins in molecular biology, Wiley aad Sons, NY (1987}, Nucleotide sequence determination by chain termination method? has also been published [Ausubei et al. (1987}). Restriction enzymes were purchased from New England Biotechnology (Beverly, MA). To perform the ligations, DNA magnets were incubated in a buffer containing a mixture of 5 mM Tris-HCl (pH 7, -¾ 10 mM MgCl), 10 mM DTT, 2 mM ATP, and tag T4 DNA ligase (Biolahs). Oligonucleotides were synthesized by phosphoramidite reaction, protecting the β position of the phosphoramidite with a cyanoethyl group [Stuka et al,. NucL Acids Rss. J2, 4539-4557 (1984); Giles, Am. Bioechnol. Nov / Dee (1985)) using a Biosearch 8600 automated DNA synthesizer according to the manufacturer's instructions. Ligated DNAs or RN8s to be tested for their transformation efficiency are used to transform the following competent strain: E. «kV DH5a[F / endAt, bsdR17, supB94, thi-1, rcc.Al, gytA9ó, relAi, AflacZY A~aru F)G 169. dsoR, ibSódlnctlaeZAMió)] (for any ColEl plasmid); or £'. euri XAC-pir (for any pCÖR-derived plasmid). Mini-preparations of plasmid DNA were prepared according to Klein et al. [Klein et al., (1950)j, LS medium is used for the cultivation of E. coli strains [Maniatis et al, (1982)]. The strains are incubated at 37°C. The bacteria are plated on plates prepared with LB medium supplemented with antibiotics. 1.1, Making the column Acmvr&zfo The column we used was a 1 ml BiTrap column activated with NHS (N-hydroxysuccinamide; Pharmacia) and connected to a peristaltic pump (output < 1 tnl / mm). The specific oligonucleotide used contained an amino group at the 5'-end and had the following sequence: 5'GACGCTTCTTCTfCTTCTrCTTCTT-S' (SEQ ID NO: 1). In this example, we use the following puffs: Switching buffer: 9.2 M NaBCC 0.5 M NaCl, pH 8.3; Buffer A: 0.5 M ethanediamine, 0.5 M NsCl, pH' 8.3; Buffer B: 0.1 M acetate, 0.5 M NaCi, pH 4, 'Wíiífeze? The column is washed with 6 ml of 1 mM hydrochloric acid and the oligonucleotide (50 nM in 1 ml) diluted with coupling buffer is applied to the column and left for 30 minutes at room temperature. The column is washed three times successively with 6 ml of buffer A and then with 6 ml of buffer B. The oligonucleotide is thus covalently bound to the column via a C-OH bond. The column is stored at 4 °C in a mixture of PBS and 0.1% NaN3 and can be used at least four times. 1.2, Plasmid generation The following two oligonucleotides were synthesized. 4817. oligonucleotide: S'GATQAVAáGáAGÁAGAáGAÁGAÁGAáGAáGAí\GAáGáAGáAG,ÁM3áőGAAGAAGAáG0G' (sequence number 9); 481 S. oligpsukfeödd: SAATKXTFimCFKniCöaiOT number sxavencta). These oligonucleotides, when hybridized and cloned into plasmids, introduce a homopurine-homopyrimidine sequence (GAA>}7(sequence number 33) into the appropriate plasmid as described above. The sequence corresponding to these two hybridized oligonucleotides was cloned into the complex cloning site of the ampicillin resistance gene-carrying pBKS+ plasmid (Stratagene Cloning System, La Joiia, CA). For this purpose, the oligonucleotides were hybridized as follows: 1 µg of these two oligonucleotides together. was placed in 40 ml of a final buffer consisting of 50 mM Tris1Ό (pH ?.4), 18 mM MgCl>. This mixture was heated to 95 ~C-rs, then placed at room temperature. so that the temperature slowly decreased. 18 ng of the hybrid oligonucleotide mixture were ligated into 280 ng of pBKS-i-pbzmidsl (Sirstagene Cloning System, La Folia, CA) and digested with restriction enzymes IrHl and EeoRl in a final volume of 30 µl. After ligation, a small aliquot was transformed into DHSo. The transformation mixtures were plated on L medium supplemented with amphiphilin (.50 mg / l) and X-gal (<20 mg / l).Recombinant clones should be free of blue color on this medium, unlike the grape plasmid (pBKS*X ansely allows £, co / r β-galactostase . <s fragmentumának ö-kompiemeniációját. plazmái dns 6 klánból történő mini preparációját követően ezek mindegyike mutatta a pbks * plszmkl eeorf és rsvííhi helyei között elhelyezkedő esd hely eltűnését az összetett klónozó helyet tartalmazó 44s bp pvuii sáv molekulatömeg növekedését. kiválasztottunk egy kiónt megfelelő plazmidot pxl25ő3 jelzéssel láttuk el. a klónozott szekvenciát szekvunálássai igazoltak, prsner-28: 5'tga.ccggcagcaaaátg-3' (11. szátnü szekvencia) (yiera and messing, gone .19, 259-268 (1982)1 alkalmazásával, puc plazraídok m13mp7-ből származó rendszerek pb.rs+ plazmld (síratagene cloning system, la jölla, ca) inszereíős matggeneziséhez szintetikus univerzális prímerekkel szekvenálásához. pxl2563 plszmídoí wizard megaprep kh (prosnega corp. madison, wí) segítségével tisztítottuk gyártó utasításai szerint. ezt plazmid preparátumot használtuk azstán alábbiakban leírt példákban. 1,3, plazmld tisztítás az 1,2, fejezetben ll oiigöoukleoíiddal kapcsolt hitrsp oszlopon & pbks+ is oldatból. tisztításhoz használt pufeerek kővetkezők voltak: f puffer: 2 m nacl,. 0,2 acélát, ph 4,5-5;: b 1 tris-hcl, 9,8,5 t«m edta. mddsjár áz oszlopot ml puíferrel mossak, piazmidokat (20 ng 20 pg pbks* 400 μΐ e pufferben) felvisszűk oszlopra érán keresztál szobahőmérsékleten inknbáljuk. 18 tűi púderrel mossák azután pufferrel eluáijuk. plasmidokas % agaróz gélen végzett elektrofbrézist etídíum-bromidos festést kővetően mutatlak ki. plazroidofc aranyát oldatban a. ooő transzformáló aktivitásuk mérésével becsüljük meg. amőkífey 30% 7'®% plazraid keverékből kiindulva 100% tartalmú oldatot nyerünk oszlop alján.. .268 mn-ea 2sö ras-es mért optikai denzitássai (od-vel) becsült tisztaság 1,9 2,5 kőzötlö ti, amely azt jelzi, hogy szennyező fehérjék eltávozlak ezzel taódsssetrel ílsáma 2.1, ez példa tisztítási kísérletet mutat he. ólígoouklsotid [5’oágöc‘fi‘cttctfcttcttcttctt-3 (1. számú szekvencia}] hozzákapcsolását az: oszlophoz 1. példában leírtak szerint végezzük. kapcsoláshoz módosítjuk oligohukieo-tídöt 5'-végeit, amkso csoportot kötünk a. hézag foszfát csoportjához s szénatomot kar (modified oiígootjcleotlde earogertíee sa, belgium), piazntidot wlzard kit (promega corp, wi) segítségévei tisztítottak ebben következő pufíereket feasztíáljuk: puffét: naci, sóstót 4,5-5; puffét; i tris-ηά, 9, 0,5 tnjm pafferrel 1öö p.xl25ó3 plsz-midoi pl pufferbett hígítva felvtszünk órán keresztül hskubáljuk. lő pufferrei pufiméi elnáljak. mennyiségét 260 ren-ea denzitás méréssel határozzuk példában: kötést olyan pofférben végezzük, amelynek molaritása nacl-ra 0-2 közötti (f puffét). tisztítás; hatásfok» csökken, ha csökken nacl molaritása. kapcsoló puffer ph-js 4,5 5 közölt változhat, hatásfoka jobb értéknél. másik, házlkus ph-iá slúeiós puffét· ís alkalmazható: ilyeníörmán elóciót végeztünk 50 mm borát edta keverékével, 2.2. oligonukleotid [5'-gaggcttcttcttcttctrctivn'-3' (1, szekvencia)] pxl25ó3 plazmldot wizsrd (frotnega puffét eket használjuk: 0,1 naci, acstáí, 5; iris-hci, 11,5 ó 100' pafferben te!viszünk az. koreszíül ínkubáijuk. az. 10 mossuk eináljnk. mintákban jelen levő £. sah genomiális vagy kromoszómáid tartalmai mérjük ohgooukieotiddal való átáramolíaíás előtt után. étinek dnsnek ecr-reí meg, primereket alkalmazva £, cetfi galk génben, következők járunk ezeknek printereknek szekvenciáját: 5''-ccgaati'cl'ggggaccaaaöcagtttc-3' (24. szántó 5'-ccaagctrcac'fgr{'cacgacgggtgt-3' (25. defeoack munkatársai írják le |nucl. acids rés. 13. 1341-1853 (1935)1 reakelóelegy 25 pcr püffedte» francé, charbosníéres) 1,5 tnm mgcb-ot, flxtp-t (efearmacía. örsay), μμ prímért .e 'ml tag pölimerázt (eromega) tartalmaz. reakciót sorrendben végezzük: perc 956c-»n; ciklus másodpere 95!>C, seconds at 6Ö °C, I minute at 78 C; min 78 °COU, ιι The amplified 124 bp DNA fragments were separated by electrophoresis on a 3% agarose gel in the presence of SybrGreen I (Molecule Profees, Eagene, USA) and then quantified by digestion with an Ultrapor generic DNA sequence (Sigtna, ref D4889) from strain E. coli B. Before loading onto the column, the sample contained 1% chromosomal DNA, while the sample purified on the oligonucleotide column contained 0.2%, Experiment 1.5 ml of a single-pass culture of the DH5a strain containing the plasmid pXL2563 is centrifuged and the pellet is resuspended in 100 μΐ of a mixture of 50 mM glucose, 25 mM Tris-HCl, pH 5 and 10 mM disodium phosphate. 200 μΐ of a mixture of 0.2 M NaOH, 1% SDS is added, the tubes are inverted to mix, then 150 μΙ of 3 M potassium acetate (pH 5) is added and the tubes are inverted to mix, After centrifugation, the supernatant is recovered and applied to the plate as described in Example 1. onto an oligonucleotide-coupled column. Binding, washing and elution are the same as described in Example 1. Approximately 1 pg of plasmid is recovered from 1.5 ml of culture. The resulting plasmid, analyzed by agarose gel electrophoresis and ethidium bromide staining, appears as a single strand of "supercoiled" circular DNA.The plasmid purified by this method contains no trace of large molecular weight (chromosomal) DNA or RNA. The ratio of optical densities measured at 260 nm and 280 nm is greater than 2. 4.1 This example describes a method for purifying plasmid DNA under the same conditions as in Example 3, from a 20 ml culture of the DH5α bacterial strain containing the plasmid pXL2563. The cell pellet is taken up in 1.5 ml of a mixture of 50 mM glucose, 25 mM Tris-HCl, pH 8 and 10 mM EDTA. The lysis is carried out in 2 ml of a mixture of 0.2 M NaOK, 1% SOS and neutralized with 1.5 ml of 3 M phthalic acid acetate (pH 5). The DNA is then precipitated with 3 ml of 2-propanol and the pellet is resuspended in 0.5 ml of a mixture of 0.2 M phthalic acid acetate (pH 5) and 0.1 M NaCl and then applied to the oligonucleotide-coupled column obtained as described in Example 1. Binding, washing and elution are the same as those described in Example 1, except for the. washing buffer, the molarity of which is Θ.Ι M for NaCl. Approximately 100 pg of plasma DNA is obtained. The resulting plasma, analyzed by agarose gel electrophoresis and ethidium bromide staining, appears as a single band of "sapper-protected" circular DNA.The plasmid purified by this method also contains no trace of small molecule (chromosomal) DNA or RNA fragments. Restriction enzyme digestion of the plasmid gives a single band at the expected molecular weight of 3 kilobases. The protein concentration in the samples decreases from 125 pg / ml measured in pure BCA to less than 1 ng / ml in the purified plasmid (Micro-BCA Assay, Pierce). The endotoxin concentration measured by LAL determination (LAL Assay, Biosepra) is divided by a factor of more than 10 in the purified plasmid compared to the starting pure lysate. 4.2. The plasmid used contains a cassette containing the cytomegalovirus promoter, the luciferase coding gene and the bomopurine-homopyrimidine sequence (GAA / ri (SEQ ID NO: 33) from plasmid p.XL.2563. The DH1 strain containing this plasmid [Manilátís et al., (1989)] is cultivated in a ? liter fermentor. A pure extract is prepared from 2Ö0 g of cells: the cell pellet is taken up in 2 liters of 25 mM Tris, pH 6.8, 50 mM glucose, 10 mM EDTA, to which 2 liters of 0.2 M NaOH, 1% SDS are added. The lysate is 1 »"· «ох<« . « » » * ν< »Χχ χ#, * " " » ·« «« «»» «. neutralized by adding 3 μL of potassium acetate. After dialysis, 4 ml of this filtrate is applied to a 5 ml Hiltop-NHS column to which the oligonucleotide with the sequence 5'GAGGCTTCTTCTTCTrC'rrcrfG'r'fCTr-3' (SEQ ID NO: 1) is attached as described in Example 1.1. Washing and elution are carried out as described in Example 1. Approximately 40 pg of plasmid are recovered. The genomic DNA content in this extract — achieved by the technique described in Example 2.2 — is 0.1%. Modified oligonucleotide: application This example describes the synthesis of an oligonucleotide containing intercalated cytosines. The oligonucleotide sequence is as follows: 5'-GAÖG^CTI^CTT^Cn'^CTTM'CTT^CTl^'8Crr-5' (SEQ ID NO: 12), This oligonucleotide contains an amino group at the 5'-end, MeC ~ 5-methyl-cytosine. This oligonucleotide allows the purification of the pXL2563 plasmid under the conditions described in Example 1, with 5 µl coupling buffer (thereby reducing the risk of plasmid degradation). In the above examples, the oligonucleotide used is modified at the 5'-end with an amino group, which is linked to the phosphate group via a 6-membered arm (NH2-(CH2)S). In this example, the amino group is linked to the 5'-terminal phosphate via a 12-membered arm (N1L-(Cl-Ι7.)t;:). The coupling of the oligonucleotide and the flow through the column are carried out as described in Example 2 with buffer F (2 M NaO, 0.2 M acetate, pH 4.5). This oligonucleotide allows for a better purification efficiency, a yield of 5354 is obtained, while with the oligonucleotide containing the 6-membered arm under the same conditions this yield is 45%. Following the cloning strategy described in Example 1.2, two other plasmids carrying the homopurine-horeoptrimide sequence were constructed: plasmid pXL2725, whose sequence is (GGA)}e (34, target sequence) and plasmid pXL272o, whose sequence is (GÁ)25 (35, target sequence). 7.1, Composition of plasmas Plasmids pXL2725 and pXL2726 were constructed similarly to plasmid pXL2503 according to the cloning strategy described in Example 1.2, using the following oligonucleotide pairs: 5986:5'-GATCC(GA>ssGGG-3' (sequence number 13), 5987:5 -AATTCCC{TC)2sG-3'' (frequency number 14), 5981: 5'~GATCC(GGA}!2OG~3' (sequence number 15), 5982t5'-AATT(CCT)t7CCG-3' (SEQ ID NO: 16). Oligonucleotide pairs 5986 and 5987 were used to construct plasmid pXI,2720 by cloning the oligomers into the &ssw'Hl and EcnRl sites of plasmid pBKS t (Strstagerse Cloning System, La Tolla, CA), while oligonucleotides 5981 and 5982 were used to construct plasmid pXE 2725. The same experimental conditions were used as for the construction of pXI,2563, except that the oligonucleotide pairs were exchanged. Similarly, the cloned sequences were confirmed by sequencing of the plasmid. This shows that plasmid pXL2725 contains a modification compared to the expected sequence: instead of the 17-fold repeated GGA sequence, GG?áGA(GGÁ)ss (SEQ ID NO: 17). 7.2, Preparation and cleaning of columns Oligonucleotides forming triplexes with these homoprotein sequences were ligated to HiTrep columns according to the technique described in Example 1. The oligonucleotide sequence 5'-AATGCCTCCTCCTCCTCCTCCT-3'08, sequence 19) was used to purify plasmid pXI.2726. The two columns thus obtained allow the purification of the corresponding plasmids according to the technique described in Example 2 with the following purines; Buffer F: 2 M NaCi δ, 2 M acetate, pM 4.5 This buffer is 1 M Tris-HCh pH 9.0.5 mM EDTA. The yields obtained were 2.3% and 31%, respectively, for pXL2725 and pXL2720. This example illustrates the influence of the length of the specific sequence present in the plasmid on the efficiency of purification. 8.1, Assembly of plasmids The reporter gene used in these experiments to indicate the activity of the compositions of the invention is the gene encoding luciferase (Lee). Plasmid pXE2021 contains a cassette that includes the cytomegalovirus (CMV) promoter, excised from p.DNA3 (Promega Corp., San Diego, CA) with the restriction enzymes M»f and Ahozdll, cloned upstream of the gene encoding the inhibitor into the restriction sites of the pGL basic vector (Promega Corp., San Diego, WI). This plasmid was constructed using standard molecular biology techniques. The plasmids pXI..2?2?" l and pXL2727-2 were assembled as follows: Plasmid p.Xl-2021 was digested with ZforsHI; the enzyme was activated by a 10-minute incubation on bSeC; during this time, the nucleotides 6008 and 6008 were hybridized as described for the construction of plasmid pXL2563. 6006: 5'-GATCT(ŰAA)!7CTGCAGATCT~3' (sequence number 20) 608: 5'>GATCAGATCTGCAG(TTC)í7A-3' (SEQ ID NO:21). This hybridization mixture was cloned into the Eoml-fl ends of plasmid pXL2621 and after transformation into DH5 strain, recombinant clones were identified by Fart restriction enzyme analysis, since the tsligonucleojds introduced a jRsrt site. Two clones were selected and the nucleotide sequence of the cloned fragment was aligned using primer 6282 (S'\ACAGTCATÁÁGTG€GG€GACG-3'; SEQ ID NO: 22) as a sequencing reaction primer [Viera and Messfog, Gene 19, 259-268 (1982)], The pDC plasmids were used for disassembly of the MÍ3mp?-derived systems for mutagenesis and sequencing with synthetic universal printers. The first clone (pXL2?2?~l) contains the GAAA sequences in 1 ö-szsrss repeat, The second (pXL2727-2) contains the sequence: 5'GAAGAAGAG(GAA)A7GAAGAG.AA-3' (SEQ ID NO: 23). 8.2, Preparation and cleaning of columns A column as described in Example I, coupled to the oligonucleotide with the sequence 5'-AGGCTTCTCTCTGTTCTTCTTCTT-3' (SEQ ID NO:1). Plasmid pXL2727-1 carries a 14-fold repeat of the GAA sequence. The oligonucleotide described in the text, which contains a 7-fold repeat of the corresponding hybridization sequence CTT, is therefore able to hybridize to the plasmid at 8 different positions. In contrast, plasmid pXL2727-2 has the same hybridization sequence (GAA) (SEQ ID NO: 3) as the oligonucleotide attached to the column. This oligonucleotide is therefore only able to hybridize to pXL2727-2 at one position. The experiment is the same as described in Example 2, with the following buffers: buffer F: 2 M NaCl, 0.2 M cetyl acetate, pH 4.5 buffer E: 1 M Tm-HCl, pH 9, 0.5 Λ ED'í'A, The purification yield was 29% for plasmid pXL2727-1 and 19% for plasmid pXL2727-2. 8.X Mammal: The cells used are NIH 3T3 cells, which are inoculated one day before the experiment into 24-well culture plates at a cell concentration of 5000 cells per well. The plasma is diluted in 150 mM NaCl and mixed with lipofedanf .RPR1 15335. The ratio of lipofedast positive charges to negative charges used is 6. The mixture is shaken, left to stand at room temperature for 19 minutes, diluted in peppermint serum-free medium and then added to the cells at a ratio of 1 µg DNA per well. After two hours at 37 °C, a volume of fetal calf serum was added and the cells were incubated for 48 hours at 37 °C in the presence of 5% CQ. The cells were washed twice in PBS and the luciferase activity was measured according to the protocol (Promega kit, Promega Corp., Madison, WI) with a Lumat LB9501. Ittmometer (£0 and G Bertfeld, Evty). In Example 82, purified p.Plasmid XL2727-1 produces twice the transfection yield as a version of the same plasmid purified using the Wizard Megaprep kit (Promega Corp., Madison, WI). Purification of pCÖR-sharnjns plasmids The following example illustrates the purification of pCOR-derived plasmids by triple helix affinity chromatography. We have already shown that this technology removes nucleic acid contaminants (mainly host-derived genomic DNA and ENS) to a level that could not be achieved by conventional chromatographic methods. Triplex affinity gels were synthesized using Sephaeryl S-1000 SF (Emmersham-Pharmacia Biotech) as the chromatographic matrix. First, Sephaeryl S-1000 was activated with sodium metaperiodate (3 mM, room temperature: 1 hour) in 0.2 M sodium acetate (pH 4.7). Then, the oligonucleotide was coupled via its S'-terminal amino acid to the aldehyde group of the activated matrix by reductive amination in the presence of ascorbic acid (5 mM), as previously described for coupling proteins [Horssey et al., J. Immunol Methods 93, 83-88 (1986)]. The homopyrimidin oligonucleotide used for these experiments had a sequence that was complementary to the short, 14-membered fetnopurin sequence (5'-AAGAAA.A.AAAGAA-3') present in the origin of replication (oriy) of the pCOR plasmid (SEQ ID NO: 29) [Soubrier et al., Ges. 6, 1482-1488 (1999)], as described above, the sequence of the abömop2maat&ohgosnkleoíid is 5'-TTCTTTTTTTTCTT-3' (SEQ ID NO: 39). The following plasmids were chromatographed: pXL3296 (pCOR without transgene, 2.0 kbp), pXL379 (pCOR-FGF, 2.4 kbp), pX1389 (pCOR-VBÖFB, 2.5 kbp, pX1389 (pCOR-AFP, 3.7 kbp), pXL3222 tpCORiaeX, 5.4 kbp) and pXL3397 (pCOK-Bdeliefed FViö, 6.6 kbp). Each of these plasmids was purified by two steps of anfonosere chromatography from the pure lysates obtained as described in Example 4. Plasmid pSKS-v (pBluescript 1 KS+, Síratagene) derived from ColEl, purified by ultracentrifugation in CsCl, was also studied. The majority of the piasrides used were in a supercoiled (> 95%) topological state. In each plasmid DNA purification experiment, 300 pg of purified DNA in 6 ml of 2 M NaCl, 0.2 M potassium acetate (pH 5.0) was applied to a column containing the aforementioned oligonucleotide (5'TTCTTTTTTT3' (SEQ ID NO: 30) at a flow rate of 30 cm / h. The column was washed with 5 volumes of the same buffer and the bound plasmid was eluted with 1 M Tris-HCl. 0.5 mM EDTA (pH 9.0) and quantified by UV (260 nm) and ion exchange chromatography on a Millipore Ösn-Fttk column [Margóet et al., SiaPhacm a, 26-37 (19951). Plasmid recoveries in the -collected fraction contained the following: 207 pg pXE329õ, 196 ug pXÍ3179, 192 ug pXO579, 139 pg pX.t.3678, 97 pg pXL3227 and 79 ug pXL3397, No Oidtank plasmid binding indicators (<3 pg) were observed when pKBS-r plasmids were chromatographed on this column. This indicates that the oligonucleotide with the sequence 5'TTCTTTTTTTTCTT-3' (SEQ ID NO: 38) formed a stable triptex structure with the complementary 14-member sequence 5'AAGAAAAAAAAGAA-3' (SEQ ID NO: 29) present in pCGR-heo (ref. / ?), but not with the closely related sequence 5'AGAAAAAAAGGA-3' (SEQ ID NO: 27) present in pBRS-beo. This indicates that the introduction of a unique non-canonical insertion (in this case T*GC) results in complete destabilization of the triptex structure. As a control, no plasmid binding was observed (<1 µg) when plasmid pXL3179, which was synthesized under strictly similar conditions but without oBgonkleotM, was chromatographed on a blank column. By running this affinity purification column between the cycles described herein, the level of contaminating host genomic DNA was reduced from 2.6% to 0.87% for pXL32.96. Similarly, contaminating host DNA was reduced from 0.539 to 0.50% for pX1.3179 when the sample was chromatographed on the same affinity column. Furthermore, the level of contaminating RNA was greatly reduced from 43% to 0.2% RNA when pXL3379 was prepared using this affinity column. In addition, the recovery of plasmid pX.L3579 was less than 8% when the 5'-TTCTTTTTTTTCTT3 (SEQ ID NO: 30) oligonucleotide was replaced by the 5'-TTCTTTTTTTTT3 (SEQ ID NO: 33) oligonucleotide on the affinity column. Although the oligo with SEQ ID NO: 31 is complementary to a portion of the VEGFB sequence present in plasmid pXL3S79 (i.e. nucleotides 379-389 relative to the ATG), significant triplex cleavage is still produced, indicating that a random homo-homopyrimidin DNA sequence is not required for this affinity purification. Example 16 Purification of the ColR1-saffron plasmid The following example illustrates the purification of ColB1-derived plasmids using triple helix affinity chromatography. It has been demonstrated that this technique removes nucleic acid contaminants (mainly host-derived genomic DNA and RNA) to a level not achievable by conventional chromatography methods. A triplex gel was synthesized by attaching the oligonucleotide 5-THIΤΙΤΠΤΓΤΟΕΤ-0' (SEQ ID NO: 28) to a periplasmic oxidized Sephacryj S-1808 SF column as described in Example 9. The plasmid pXI.3296 (without p€ÖR insertion) and the GolEl-derived pBKS were chromatographed on a 1 ml column containing the 5'· TCTTTTTTTCCT-3'' (SEQ ID NO: 28) oligonucleotide under the conditions described in Example 9. The recovery of the plasmids was carried out as follows: 175 ug of the plasmid was collected. pBKS and < 1 pg pXL329ő, This indicates that the oligo-nucleotide $'-'ΓΟΠΤΠ?Τ€€Τ-3><SEQ ID NO: 28) forms a stable triplex structure with the complementary 12-membered sequence present in pBKS <5áGAAAÁÁAAG <jA-3‘; 27. számú szekvencia), a pCOR-ben jelen levő, ezzel sjww rokon 12-iagú 5'« AGAAÁAAAAAGA-3 j :(32.. számú szekvencia) szekvenciával viszont nem. Ez azt jelzi, hogy egyedi nemkanonikus íriád (ebben az esetben C*ÁT) bevezetése a íriplex szerkezet teljés destabili-zácíóját eredményezi. The following example illustrates the purification of a supercoiled double-stranded DNA molecule, such as pX.1.3296, from a triple helix affine solution containing another double-stranded DNA molecule, such as pBSK. Both double-stranded DNA molecules are of similar size, but each DNA molecule contains a unique sequence that is capable of forming a triple helix with a different target sequence. As previously described, a molecule, such as pXL3296, contains the sequence 5'-AAöAAÁAÁ.AAÁC?ÁA~3' (SEQ ID NO: 29), but does not contain the sequence 5'-AGAÁAAAAAGGA-3' (SEQ ID NO: 27). In contrast, molecules such as pBRS contain sequence number 27 but not sequence number 29. The solution was passed through the first column, which contained unbound DNA molecules. In the second step, unbound DNA molecules were passed onto a second affinity column, which contained the oligonucleotide 5-TrCTTTTTTTTTCT~3,:(SEQ ID NO:30), such as the column described in Example 9. The second column was then washed and the bound molecules were recovered from the second column as described in Example 9. Only pXL329o molecules eluted from the second column, no pBSK molecules were detected from the second column effluent (i.e. from the solution that came off the column). 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Lessons of ίοε&σ: <fe&fe ttettá 38 ítífca <6 <21022 <2H>21 <2Í2>DNA <21.0' Fairy tales <©> 220 <12> DNA <213> Mssteéges sas&vencia <220 <220 Description of the mességes vckveseía: színSsfes digíwkfeo&l «023 Ss^g^a^segssEss^agaagaagaaggaagagfSa 39 <21024 <21027 <12> DNA <210 Artificial smOwesa <20 <220 Description of the asesteségs saáomgs: sártókus digix&ékööá •Φ φ * « * φ φ * φ ΦΦ φ#* « « * « X φ ♦ Φ * ♦ φφφ XX <40024 ccgaaStíg eggsxaaag ca@®e <25025 <25Ι>27 <212> DNA <220 -323> Description of the KK-s&afegso sssqvence: sattóas ofeoiásleoüd «5025 <xaagdJeacígteac^c^gS <31026 <211> 21 <2;2>öNA <213> MesaOges xOveicia <220 <223> The sequence of the written text is: saníeSiks GfemskleNíd <40026 <5MKSCtXSCit3t 21 <21027 <21012 <2!2>DNA -313> Artificial sadforaxás <220 '320 The msstö'séges ssátvencá: feaMc ♦ RX * <4002? aggaaaaaaaagg 12 <21028 <21012 <212> DNA <213> Mss&séggs saáámás <223> ? <40028 «21029 «210 14 <212> DNA <210 Mestsrtásssssssvweb <220 «220 The mes&ssségss sequence teas: <40029 mg?saaam aggja 14 <21030 <21014 «21ΟΠΜΑ '<220 <220 A 'rtóstaségss smkvűtcá fcáása; szintósüs oligffiuádeodd <40030 ·♦ ** ♦ V * * * « χκ »»♦ » « ♦ * » * * « W ♦# AX Κ * ♦ <21031 <2ϊΊ>ίΙ <210 DNA <310 Mes&ssém <2 jsssías^s saskvaóí fefesa: SÍUfsglkysoSgsmkfotsld <40031 Wlícct 11 <21032 <2Í1>12 <212·=· DNA <210 Mesteúges saskvanda <220 <223> A iwsgfsegss sggfe wm feíbsa: x?3!3&^í£ÍöIlg,i.wkleööd <40032 sgíssssuussi 12 <21033 <21051 <212> DNA <213> Messsséges .gaEkuencsa <220 <220 A mateégss ssAvök szintóeis -oligoodfcteeöd «033 ♦ * « β ·**♦· »** <21034 <21043 <212> DNA <213> Mesteséges ssáöwcb <220 <223> The reverse sequence: srin&íikas OgmukfeoO <21035 <21050 <212> DNA <213> Möstenbgps sequence <220 <223> The mssissségss sequence is judged: synthetic oiigrtvukieo&l <40035 g!ga^g^ss^,a^a£sgag5g^agagaoagagsígaES£s^2ss 50 <21036 <21021 <212> DNA <213> Artificial .saefomási <220 <222> Á mestóisss sadcvmoaiáása; sste&«soSst8sJ <40036 »««♦ «« * * X ·'«· * β * ♦ φ * * < / h> < / s>

Claims

Patent claims 1. Process for purifying double-stranded DNA from a solution containing double-stranded DNA mixed with other components, characterized in that it comprises passing the solution through a covalently linked column of an oligonucleotide which, upon hybridization with the structural sequence present in the double-stranded DNA, is capable of forming a triple helix with the double-stranded DNA without the formation of non-canonical trilades, wherein the covalently linked oligonucleotide comprises the sequence 5'-Τ€ΤΤΠΤΠ'€ΟΓ-3' (SEQ ID NO: 28) or 5'-ΤΓ€ΊΤΠΤ'ΠΤ€ΤΓ-3' (SEQ ID NO: 30), 2. A method for purifying double-stranded DNA from a site containing double-stranded DNA mixed with other components, characterized in that it comprises passing the solution through a column covalently linked to a olgonac-leolide which is capable of forming a triple helix with the double-stranded DNA by lubricating with a six-repetitive sequence present in the double-stranded DNA without the formation of non-canonical triads, wherein the specific sequence present in the double-stranded DNA comprises the sequence 5'-AGÁAAAAAAGGA-S' (SEQ ID NO: 27) or 5'AAGAAAAAAAAGAA-3' (SEQ ID NO: 29).

3. A method for purifying a first double-stranded DNA from a solution comprising the first double-stranded DNA and a second double-stranded DNA, comprising (1) passing the solution through a first column containing a covalently bound oligonucleotide capable of forming a triple helix with said second double-stranded DNA by hybridization with a specific sequence present therein without the formation of non-canonical triads, (ii) recovering the solution passed through the first column and (iii) passing the recovered solution through a second column containing a covalently bound oligonucleotide capable of forming a triple helix with said first double-stranded DNA by hybridization with a specific sequence present therein without the formation of non-canonical triads, wherein the specific sequence present in said first double-stranded DNA sequence reads 5- / 1AGAAAAAAAAGAA-3' (29.(SEQ ID NO: 27) and the specific sequence present in said second double-stranded DNA comprises the sequence 5'-AGAAAAAAAGGA'3! (SEQ ID NO: 27).

4. A method for purifying a first double-stranded DNA from a solution containing a first double-stranded DNA and a second double-stranded DNA, characterized in that it comprises (1) passing the solution through a first column containing a covalently bound oligonucleotide capable of forming a triple helix with said second double-stranded DNA by hybridization with a specific sequence contained therein, (ii) recovering the solution passed through the lower column and (iii) passing the recovered solution through a second column containing a covalently bound oligonucleotide capable of forming a triple helix with said first double-stranded DNA by hybridization with a specific sequence contained therein, wherein said first double-stranded DNA forms a triple helix with said first double-stranded DNA. The oligonucleotide capable of forming contains the sequence ω'-TTCrnTiTTrClT-S' (3ω.sequence) and the sequence capable of forming a triple helix with said second double-stranded DNA comprises the sequence 5TC'rrrnTFCC Γ-3' (SEQ ID NO: 28). <· * $ * « ** *«* ♦ « * * * * r*« «» #♦* **.

5. The method according to any one of claims 1-4, characterized in that the cell is 6. The method according to claim 5, characterized in that the cell lysate is a pure lysate.

7. The method according to any one of claims 1-6, characterized in that the double-stranded DNA is previously purified.

8. The method according to any one of claims 1-7, characterized in that the specific sequence is artificially introduced into the double-stranded DNA, but is naturally present in the double-stranded DNA.

9. The method according to any one of claims 1-8, characterized in that the oligonucleotide is attached to the column via a disulfide, isoether, ester, amide or amine bond, 10. The method of claim 9, wherein the oligonucleotide is attached to the column by an arm containing a carbon (CH2)3, wherein n is a number between 1 and 5.8 (inclusive) and wherein the arm is attached to the oligonucleotide via a phosphate bond and to the column via an amide bond.

11. The method according to any one of claims 1 to 11, characterized in that the oligonucleotide has at least one chemical modification that renders it resistant to or protects it from nickases or increases its affinity for the specific sequence.

12. The method according to claim 11, characterized in that at least one nucleotide of the oligonucleotide is methylated.

13. The method according to any one of claims 1-12, characterized in that the double-stranded DNA is circular DNA.

14. The method of claim 13, wherein the circular DNA is a plasmid.

15. The method according to any one of claims 1-14, characterized in that the specific sequence present in the double-stranded DNA contains a plurality of positions for hybridization with the oligonucleotide, 16. The method according to any one of claims 1-15, characterized in that the support is a functional chromatography column, a functional plastic surface or a functional teex bead.

17. The method of claim 10, wherein the support is a functional chromatography column.

15. The method of claim 17, wherein the purified double-stranded DNA has a chromosomal DNA content of 0.596 or less.

19. The method of claim 18, wherein the purified double-stranded DNA has a DNA content of 0.01% or less in the chromosome.