MXPA99003239A - Methods for labeling nucleotides, labeled nucleotides and useful intermediates - Google Patents
Methods for labeling nucleotides, labeled nucleotides and useful intermediatesInfo
- Publication number
- MXPA99003239A MXPA99003239A MXPA/A/1999/003239A MX9903239A MXPA99003239A MX PA99003239 A MXPA99003239 A MX PA99003239A MX 9903239 A MX9903239 A MX 9903239A MX PA99003239 A MXPA99003239 A MX PA99003239A
- Authority
- MX
- Mexico
- Prior art keywords
- separator
- chain
- dna
- nucleotides
- reactive
- Prior art date
Links
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- 229940111202 pepsin Drugs 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000001376 precipitating Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 150000003141 primary amines Chemical group 0.000 description 1
- 230000002035 prolonged Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000002285 radioactive Effects 0.000 description 1
- 238000000763 radioactive isotope labeling Methods 0.000 description 1
- 239000003638 reducing agent Substances 0.000 description 1
- 108091007521 restriction endonucleases Proteins 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
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- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
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- 238000010561 standard procedure Methods 0.000 description 1
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- 239000001226 triphosphate Substances 0.000 description 1
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Abstract
The present invention provides improved methods for labeling nucleotides. The method of the invention for labeling nucleotides comprises the steps of:reacting a reactive moiety ofa linker, which linker is a platinum compound having a stabilizing bridge and two reactive moieties, with an electron donating moiety of a spacer, which spacer comprises a chain having at least four atoms and at least one heteroatom in the chain, which spacer further comprises said electron donating moiety at one end of the chain and a reactive moiety at the other end of the chain;reacting the reactive moiety of said spacer with a label;reacting the other reactive moiety of said linker with a nucleotide. A major advantage of the invention is that all nucleotides can be labeled by the method of the invention, whereas until now the attachment of a label was mostly restricted to one or certain nucleotides.
Description
METHODS FOR MARKING NUCLEOTIDES, MARKED UCLEOTIDES AND USEFUL INTERMEDIARIES
DESCRIPTION OF THE INVENTION The invention relates to methods for labeling nucleotides using binders (linking portions between bioorganic labels and molecules, linkers which are based on platinum compounds). Platinum (coordination) compounds have been considered interesting molecules for a long time. For a review of these compounds and their uses, we refer to Reedijk et al. (Structure and Bonding 67, pp. 53-89, 1987). Cis-platinum has especially received a lot of attention as a possible antitumor drug. This antitumor reactivity of the platinum compounds originates from having at least two reactive groups (preferably in cis orientation with each other), which makes it possible for them to cross-link DNA molecules, thus inhibiting the duplication of those DNA molecules. British patent application 2 148 891 describes cis-platinum complexes, which are coordinated in position six. Platinum is bonded to two halogens or hydroxy groups, two additional halogens and to a group derived from ethylene diamine, such as 1,2-diamino-2-
REF .: 29889 methylpropane or l, 2-diamino-2-methylbutane. It is said that the complexes have an excellent antitumor effect. In the European patent application, platinum complexes coordinated in the four position with 2, 3-alkyl-1,4-butanediamine and two halogens are described for their antitumor effect. Different platinum complexes coordinated in position four are described in European patent application 0 386 243. The complexes comprise a bidentate ligand of diamine and two 2-alkanoic acids or ligands of 3-aryl-2-oxoalkanoic acid. It is established that these complexes have a strong inhibitory action of growth on certain leukemic cells and are used for their oncostatic activity. U.S. Patent No. 4,207,416, discloses ethylene diamine-platinum (II) 2,4-dioxopyrimidine complexes having high antitumor activity and low toxicity in mammals. A different use of the platinum (coordination) compound has been described in the PCT application (WO92 / 01699), wherein a platinum compound having only two reactive portions (referred to as leaving groups there) is reacted with a fluorescein to obtain a labeled platinum compound, which can bind (non-covalently) to a nucleic acid, preferably, at the N-7 position of the guanine residue. Several methods for nucleotide labels have been described in the literature. For a long time, the standard method has been to use radioactive isotope labeling. However, there are numerous problems associated with the use of radioisotopes, such as potential health risks, elimination problems and instability problems. To overcome these problems, Dale et al.,
Biochemistry, 14, (1975), 2447-2457, have proposed using direct covalent mediation as a technique for labeling nucleotides and polynucleotides. It was found that cytosine and uracil can be cured at their C5 position under moderate conditions. In addition, Gebeyehu et al., Nucleic
Acids Research, 15, (1987), 4513-4534, have reported that adenine and cytosine can be labeled with biotin derivatives through an aliphatic linker of 3 to 17 atoms. A major disadvantage of these known methods is that they are not suitable for marking all the different nucleotides, for example, Dale et al. Reported that their covalent mediation method leads to negative results for the bases adenine, tiin and guanine. In some cases, for example, when only a few residues of a certain nucleotide are present in a certain nucleic acid or when the terminal nucleotide residue of a nucleic acid has to be labeled, it is desirable to have a method of elimination to label any nucleotide residue. The present invention provides such a method The method for labeling nucleotides of the invention comprises the steps of: reacting a reactive portion of a linker of formula
wherein X represents any stabilizing bridge, and wherein A and B represent the same or different reactive portions, with an electron donor portion of a separator, which separator comprises a chain having at least four atoms and at least a heteroatom in the chain, separator which further comprises the electron donor portion at one end of the chain and a reactive portion at the other end of the chain; reacting the reactive portion of the separator with a mark;
reacting the other reactive portion of the binder with a nucleotide. According to the invention, the binder can be attached first to the nucleotide, and then to the separator, or vice versa, and the separator can be coupled first to the label and then to the binder or vice versa. The reactive portion of the separator can be any reactive portion that permits reaction between the separator and the label such that a marker portion comprising a label and a separator is formed, which labeling portion is sufficiently stable. The main purpose of marking nucleotides is that those labeled nucleotides can be incorporated into nucleic acid molecules. Modified nucleotides, especially those where a brand is attached
(voluminous) to the nucleotide, often they are integrated to nucleic acids with a much lower efficiency. The methods according to the invention result in the labeled nucleotides, which are integrated to the nucleic acids with a higher efficiency than the labeled nucleotides available to date. This is probably due to the selection of the separators according to the invention e? combination with the platinum-based binders according to the invention.
The mark to be used according to the invention is not critical. In principle, all brands that can bind to a nucleotide and that are used to date can be used. These labels can be radioactive labels, enzymes (which need to react with a substrate to be detected), components of specific binding pairs such as avidin, streptavidin or biotin, biocytin, iminobiotin, colloidal dyes, fluorochromes (rhumidine, etc.) , reducing substances (eosin, erythrosin, etc.), latex soles (colored), digoxigenin, metals (ruthenium), metal sols or other particulate sols (selenium, carbon and the like), dansil lysine, infrared dyes, coumarins (amino) methyl coumarins), antibodies, protein A, protein G, etc. The invention has greater benefit with the more bulky markers such as biotin, avidin, streptavidin, digoxigenin or functional equivalents thereof. The invention is not limited to nucleotides or nucleosides as such; ^ Derivatives and functional equivalents are also included. The usual nucleotides adenine, thymidine, cytosine, guanine and uridine are preferred. Especially preferred are purines, which have a good rate of incorporation. For the coupling of the separator to the platinum binder, an electron donating portion is required.
In a preferred method, the electron donor portion is an amine or a thiolate anion, both of which have proven to be very successful. It was found that aromatic amines, such as imidazoles or purines, are capable of forming very strong bonds with platinum and, thus, are well suited to be used as the electron donating portion. The separator is an important aspect of the present invention; provides an easier coupling between the marker and the binder. To avoid steric hindrance in the incorporation of the nucleotide to the nucleic acid, it should be at least four atoms in length, preferably it is at least four carbon atoms in length and has at least one heteroatom in that carbon chain. A heteroatom confers a certain amount of stiffness on the separator. That stiffness provides additional assurance that the steric factors will not obstruct a convenient linkage of a nucleotide and a tag. It is preferred that at least one hetero atom be an oxygen atom, which positively affects the hydrophilicity of the separator. Preferably, the spacer comprises no more than 20 carbon atoms in the chain, which is preferably an essentially unbranched chain, without thereby causing steric hindrance. The reason for this will be clear. A highly preferred spacer is 1,8-diamino-3,6-dioxaoctane, here referred to as Dadoo. Dadoo is a very flexible compound with a distal primary amine group and a size which makes it very suitable for use as a separator according to the invention. Another highly preferred separator of the invention is an oligolysin or a polylysine. Due to its structure and conformation, these molecules create the most convenient environment for an optimal interaction between the real brand, the nucleotide and the platinum. An additional advantage of the use of the lysine chains as a spacer is that by altering the number of units of the chain in the chain, optimal conditions can be achieved for specific brands and nucleotides or nucleic acids. Given a certain application, experts in the art will easily determine how many units of lysine are required to obtain optimum results. A particularly interesting marker portion comprising a mark and a separator has the formula
or the formula
2 NH2- (CH2) "- NH2 \ / Pt * (III) z mark '
where X represents any stabilizing bridge, Z and Z * represent a non-salient ligand and n is an integer from 2 to 10. Accordingly, the linker-separator-label system, or marker substance, with the marker portion of formula (II) ) or formula (III) has the formula
brand
or the formula
A where A, X, Z, Z 'and n have the above meanings. Non-salient ligands and / or are preferably an NH3, NH2R, NHR2 or NR3 group, wherein R represents an alkyl group having 1 to 6 carbon atoms, because those ligands have an even smaller leaving group character. than other non-salient ligands. The interesting feature of using the marker portions of formulas (II) or (III) is that both the loop and the actual label have the benefit of being directly linked to a platinum atom, while at the same time those portions are sufficiently separated to avoid steric hindrance. The binders according to the invention are preferably platinum compounds wherein X represents an aliphatic diamine. In a preferred embodiment of the invention, one or both of the nitrogen atoms of the aliphatic diamine will be protected. A suitable way to protect these nitrogen atoms consists of the blockage with one or two alkyl groups of 1 to 6 carbon atoms, preferably methyl groups. It is advantageous if the hydrogen bond between the triphosphate group of a nucleotide and the stabilizing bridge is avoided. Preferably, a diamine having 2-6 carbon atoms is used, more preferably an ethylene diamine group, which is well known for its stabilizing effect on this class of platinum compounds. In this case, the linker has the formula
H- > C CH.
G N NG. \ / Pt / \ (VI) A B
wherein G represents hydrogen or an alkyl group of 1 to 6 carbon atoms and A and B represent the same or different portions. The coupling or reactive portions A and B are preferable the same and are selected from the group consisting of N03 ~, S03 ~, Cl ", I", or other halogens. The invention of course also encompasses a labeled nucleotide obtainable by a method as described above. In addition, the invention encompasses a marker substance for labeling nucleotides by a method as described above. The marking substance of the invention has the formula 1 portion: marker
wherein X and A have the above meanings and the marker portion comprises a label and a separator as described above. Of course, the marker substances of the invention can also be used for the purpose of labeling other nucleotide labels. It was found that numerous (bio-) organic compounds, ie almost bioorganic molecules which contain an accessible sulfur or nitrogen atom, for example proteins, can be labeled with the platinum compounds of the invention. A greater advantage of the invention arises from the use of platinum compounds having the formula (I) co or linkers in the methods for preparing labeled nucleotides according to the invention. These binders can be prepared by convenient and reliable methods. In WO92 / 01699 the described starting compounds for preparing labeled platinum compounds are the
(ethylene diamine) platinum dichloride (II) and
(ethylenediamine) (Me2S0) Cl of platinum (II). The first can be obtained commercially, the second (the preferred one) must be synthesized. In the dichloride compound, the Cl ~ ions are less easily substituted by a label or a nucleotide, respectively. In the latter case, the total nucleotide labeling time may be appreciably prolonged, up to several hours, instead of several minutes. The methods for preparing the binders which are used in the method for labeling nucleotides according to the invention are based on the selection of suitable starting compounds of formula PtE4 wherein E is an electronegative group, preferably a halogen or N03 ~ or S03 ~. The reaction, which is described in the examples, of such starting compounds as for example ethylene diamine is very simple and efficient. In addition this reaction leads to very suitable symmetric intermediates to produce labeled nucleotides. A major advantage of using these compounds is that when a stabilizing bridge for the resulting platinum compound has been bound, blocking reagents need not be used. Another advantage is that the resulting intermediate compounds can be marked again without the use of blocking agents. Therefore the steps of removing the blocking agents can be completely eliminated. In addition, the yields of these reactions are very high. Yet another advantage of the use of such symmetric starting compounds is that mixtures of two different resulting compounds can not be formed, which can interfere with the next reaction and reduce the yield or require extra separation steps. A very suitable intermediate compound according to the invention is platinum (II) (ethylenediamine) (N03) 2. This substance can be easily provided with a suitable separator and a marker group, resulting in marker substances which can, through the replacement of the remaining group N03, be linked to a nucleotide very easily. In addition, the methods for producing those compounds and the resulting compounds do not involve highly toxic substances such as DMSO. Intermediates can be labeled with any suitable label (also known as a label) through an in separator as described herein above. In addition, the known advantages (of O92 / 01699 for example) are of course also obtained with the methods and compounds herein. Another advantage of platinum compounds is that they can be detected more or less directly by using platinum as a core to deposit silver or other metal crystals. By attaching the labeling substance to a nucleotide residue, single or strand or other DNA or RNA molecules can be readily detected, but it also allows the production of probes for hybridization techniques wherein the unlabeled DNA / RNA molecules are hybridize with the labeled probe. The nucleotides marked with the platinum binder do not interfere strongly with the hybridization, at all. Also, this technique obviates the use of modified nucleotides to prepare the probes. Modified nucleotides according to the practices of this invention and oligo- and polynucleotides in which the modified nucleotides have been incorporated or oligo- and polynucleotides that have been directly modified using these novel platinum compounds can be used as probes in biomedical research, clinical diagnostics and recombinant DNA technology. Other advantages and methods of the invention will become clear from the following experimental part and the examples.
EXPERIMENTAL PART
S NTESIS OF INTERMEDIARY PLATINUM COMPOUNDS These compounds, ie the binders having the formula (I), can be prepared by a process, which involves: (a) reacting a compound having the structure: with KI in a solvent suitable under suitable conditions to form an iodinated platinum compound having the structure:
(b) reacting the iodinated platinum compound with ethylenediamine in a suitable solvent to form an iodinated ethylene diamine platinum compound and represented by the formula Pt (en) I2 having the structure:
(c) reacting the compound with AgN03, the reaction is carried out in a suitable solvent, under suitable conditions to form a compound having the structure: (d) reacting the compound with KCl in a suitable solvent under suitable conditions for form a compound that has the structure:
(e) reacting the compound with. AgN03 in a suitable solvent, under suitable conditions to form a compound having the structure:
(f) recovering the modified platinum compound from the compound for the synthesis of Pt (en) compounds bound to a hapten to be used as a label for DNA and / or RNA.
Example 1 A. Preparation of the starting material Pt (en) -diamine. Preparation of Platinomethylenediamine (N03) 2: starting material. Pt (en) (N03 > 2
All reactions were carried out in the dark. Dissolve 1 gram of potassium tetrachloroplatinate (II), K2PtCl (2.4 mmol, Sigma) in 50 ml of millipore (filtered water) and stir at room temperature. Add 10 equivalents of potassium iodide, KI (24 mmol, 3,999 g, Sigma). The color of the solution will immediately turn from orange to dark red (K2PtCl, 3), stir for 5 minutes. Add one equivalent of ethylenediamine (2.4 mmol, 160.8743 μl, Merck 11 = 0.9 kg), then dilute 161 μl of ethylenediamine in 5 ml of millipore slowly with the platinum solution, mix this solution for 1 hour at room temperature. It will form < a yellow / brown precipitate, Pt (en) I2, and upon standing the previous liquid will become clear. Filter the solution through a 1.0 μm membrane filter (Schleicher &Schuell), wash the precipitate with millipore, ethanol and diether (in this order). Dry the Pt (en) I2 for at least 4 hours in a vacuum drying oven at 37 ° C. Weigh the dry Pt (en) I2 (~ 1.07g) and suspend it in 45 ml of millipore / 5 ml of acetone, the solution will be turbid, add 1.95 equivalents of AgN0 (M = 169.9, Sigma). overnight at room temperature Filter the solution through a 1.0 μm membrane filter, the precipitate is silver iodide, Agi, the filtrate should be clear Add to 0.5 ml of the filtrate, Pt (en) (N03) 2, an excess of KCl or NaCl and ensure that a white precipitate does not form immediately after adding excess KCl or NaCl. If no white precipitate formed
(only one yellow) then add an excess of KCl or NaCl to all the filtrate. After the yellow precipitate forms, filter the solution and wash the precipitate (Pt (en) Cl2) with millipore, ethanol and diether. Dry the precipitate for at least 4 hours in a vacuum drying oven at 37 ° C. Weigh the dry Pt (en) Cl2 (M = 326, l), and suspend this in 45 ml of millipore / 5 ml of acetone and stir the turbid suspension. Add 1.95 equivalents of AgN03 and stir the solution overnight at room temperature. The color of the solution will turn white, due to the formation of AgCl.
Filter the solution in the dark and evaporate the filtrate to remove the acetone by means of rotary evaporation to leave 25 ml of the filtrate. The filtrate is then lyophilized. The product is verified by NMR or Infrared Absorption Spectroscopy.
B. Preparation of starting material Pt (tmen) -diamine. Preparation of Platinum-N, N, N ', N' -tetramethylethylenediamine < N03) 2? Starting material. Pt (tmen) (N03) z
All reactions were carried out in the dark Repeat the whole process of Example 1A, but use N, N, N ', N' -tetramethylethylenediamine instead of ethylenediamine
Example 2 A.Preparation of fPt (en) (BioDadoo-M%) (N? 3? L (N03) Dissolve Pt (en (N03) 2 (18.2 mg, 0.048 mmol) in 10 ml of Miliporo water and heat until Dissolve
BioDadoo (20 g, 0.053 mmol, obtained from Boehringer Mannheim) in 5 ml of Miliporo water. Add the two solutions together and adjust the pH to 8 with 0.1 N NaOH, react for at least 3 hours at 50 ° C. Isolate the final product by lyophilization.
B. Preparation of fPtCtmen) (BioDadoo-KEk) (Q3) 1 (Q3) Dissolve Pt (t en) (N03) 2 (35 mg, 0.08 mmol) in 12.5 ml of Miliporo water and heat until dissolved. Dissolve the BioDadoo (32 mg, 0.085 mmol) in 10 ml of Miliporo water. Add the two solutions together and adjust the pH with 0.1 N NaOH, react for at least 4 hours at 50 ° C. Isolate the final product by lyophilization.
C. Preparation e fPt (en) (DigDadoo-37Ha) < N03) 1 (NQ3) Dissolve Pt (en) (N03) 2 (5 mg, 0.013 mmol) in 5 ml of Miliporo water and heat until dissolved. Dissolve the DigDadoo (9 mg, 0.016 mmol, obtained from Boehringer Mannheim) in 5 ml of Miliporo water. Add the two solutions together and react for at least 4 hours at 50 ° C. Isolate the final product by lyophilization.
Example 3 A. Preparation of a labeled dGTP Dissolve [Pt (en) (Bi? Dadoo-NH2) (N03)] (N03) (9 mg, 0.012 mmol) in 2 ml of Miliporo water. Add 2'-deoxyguanosine-5'-triphosphate (2.3 mg, 0.004 mmol) and adjust the pH to 6. Incubate for 24 hours at 50 ° C, lyophilize and dissolve in Miliporo water (1 ml) and filter through a filter of membrane. Apply the mixture to FPLC with MonoQ and purify with a linear gradient of 100% Millipore water to 100% 1M NH4HC03, collect and collect the appropriate fraction and isolate by lyophilization. Dissolve the product in a 100 mM solution of triethylamine ammonium acetate (TEAA) (1 mL) and apply to an Inverted Phase CLAP (C18) with a linear gradient of 100% 100% TEAA to 50% 100% TEAA / 100% 50mM TEAA / acetonitrile (1/1 v / v), collect and collect the appropriate fraction and remove the solvents by repeated evaporation under vacuum. Pass the product on a cation exchanger (Do ex) in lithium form, isolate the product by lyophilization.
B. Preparation of a labeled 5-AA-dUTP Dissolve [Pt (en) (BioDadoo-Mfe) (N03)] (N03) (6 mg, 0.008 mmol) in 2 ml of Miliporo water. Add 2'-deoxyuridin-5-aminoalyl-5'-triphosphate (2 mg, 0.004 mmol) and adjust the pH to 8. Incubate for 24 hours at 50 ° C, lyophilize and redissolve in Miliporo water (1 ml) and filter at through a membrane filter. Apply the mixture to an FPLC with MonoQ and purify with a linear gradient of 100% Miliporo water to 100% 1M NH4HC03, collect and collect the appropriate fraction and isolate by lyophilization. Dissolve the product in a 100 mM solution of triethylammonium acetate (TEAA) (1 ml) and apply to an inverted phase CLAP (C18) with a linear gradient of 100% 100 M TEAA to 50 mM 50% TEAA / 100% TEAA 50% / acetonitrile (1/1 v / v), collect and collect the appropriate fraction and remove the solvents by repeated evaporation under vacuum. Pass the product on a cation exchanger (Dowex) in the form of lithium, isolate the product by lyophilization.
Example 4 Reaction for coupling the Pt (en) compounds to the DNA Typical reaction for labeling DNA molecules with a Pt compound according to the invention 5 μg of double-stranded or DNase-treated DNA is sonicated to produce 300-fold fragments 500 bp 6 μg of Pt (en) compound is added and the volume is adjusted to 50 μl with demineralized water. The reaction mixture is incubated at 65 ° C for 1 hour. The unbound Pt (en) compound is blocked by adding 100 μl of a NADDTC solution. The DNA labeled with the compound of Pt (en) is purified on a sphadex G-50 column. Already labeled and purified DNA is stored at -20 ° C or used directly in a DNA probe assay. DNA probes labeled with Pt (en) compound can be stored at least 2 years at -20 ° C without loss of activity and / or specificity. All the mentioned applications are carried out with probes marked according to this protocol.
Example 5 Biotin labeling of DNA probe with EPt (en). { BioDadoo- NH2) (N03)] (NOs) (BioDadoo-ULS). Intrusion The labeling method has been used to mark DNA probes with biotin for In Situ Hybridization (ISH). In this example the marking procedures are presented, including the protocols and data for the quality control procedures. For the labeling with Biotin, total DNA cloned with Human Papilloma Virus type 6 plasmid (HPV-6, 40% GC base pairs) was used.
EXPERIMENTAL PROCEDURES Preparation of the plasmid DNA Total DNA of Human Papilloma Virus type 6 was cloned into the vector pSp-64. The plasmid DNA was transferred to E. coli (C-600) and grown on colonies of a single "LB plate containing ampicillin, where they were grown overnight in a long culture." The plasmid DNA was isolated according to the method of Birnboim and DolyX were purified by Sepharose C1-2B column chromatography (Pharmacia) and checked for inserts by restriction enzyme analysis.The concentration of plasmid DNA was determined by A260 / 280 absorption after ethanol precipitation , the DNA was reconstituted in 10 mM TRIC / HC1, pH 7.2, 0.3 M EDTA at a final concentration of 1 μg / μl (lot # 150894) .Then, this DNA was sonicated (Soniprep 150., MSE) 3 times during 10 days. minutes (amplitude of 5 microns) on ice The size of the resulting DNA fragments was determined by electrophoresis on a 2% agarose gel and found to be between 200-400 base pairs (lot # 051094).
Marking and purification of plasmid DNA HPV-6 plasmid DNA was labeled with BioDadoo-ULS by mixing the following reagents: plasmid DNA HPV-6 (lot # 051094) 5 μl (1 μg / μl) BioDadoo-ULS marker reagent 8 μl (1 μg / μl) (lot # BX940830) Demineralised water (<0.2 / μS / cm) 37 μl The 50 μl reaction mixture was incubated for 15 minutes at 85 ° C.
The excess marker reagent was captured by adding 50 μl of sodium diethyldithiocarbamate (2% solution in demineralised water) and incubating for 30 minutes at room temperature. The unbound BioDadoo-ULS was removed using a S300 HR microcentrifuge column (Pharmacia), by size exclusion chromatography. The volume of the eluent was adjusted to 500 μl, giving a concentration of HPV-6 probe with biotin of 10 ng / μl (lot # 061094).
Quality control for the detection limits The limit of detection of the biotin probes of the invention was determined by direct staining and hybridization in an inverted filter according to the following protocols:
Direct Machado The HPV-6 probe (batch # 061094) labeled with biotin according to the invention was diluted 10 times in series in stain buffer comprising 900 mM sodium chloride, 90 mM sodium acetate and 200 μg / ml of Sperm DNA from single-stranded salmon, giving a dilution series of 1000-0.1 pg of biotin probe per μl. 1 μl spots were applied on the nitrocellulose membrane and incubated for 2 hours at 80 ° C to join the DNA. The biotin probe was visualized using a conjugate of streptavidin-alkaline phosphatase (Sigma) conjugated with a precipitating substrate solution of NBT / BCIP (Sigma) according to the following protocol: - The membranes were impregnated in solution of
TES with a 0.5% tween20 (TBST) content for 5 minutes. The membranes were incubated with Strep-AP (3 DEA
U / ml) in TBST for 15 minutes at 37 ° C. - The MC membranes were washed 3 times 5 minutes in TBS solution followed by a 5 minute wash step in demineralized water. The membranes were incubated with NBT / BCIP substrate solution for 15 minutes at 37 ° C, then washed in demineralised water and air dried.
Results Using this method, it was found that the limit of detection of the DNA probe with biotin according to the invention was less than 1 pg.
Filter Hybridization, inverted HPV-6 DNA (batch # 051094) was diluted 1 in 10 in O.NN NaOH, incubated at 100 ° C for 5 minutes and placed directly on ice for 5 minutes to produce DNA with a single strand A 10-fold serial dilution in cold O.lN NaOH was made to give a series that varied from 10,000-1 pg of DNA per μl. 1 μl spots were applied on a Nylon membrane (Boehringer Mannheim) and air dried. The HPV-6 DNA probe that was labeled with biotin according to the invention was diluted in 0.5% SDS solution with 5xSSPE to give a concentration of 200 ng / ml. This solution was incubated for 5 minutes at 100 ° C and placed directly on ice for 5 minutes. The nylon membranes containing the target DNA were impregnated in 2x SSC for 5 minutes, and subsequently incubated with the single-stranded probe solution for 2 hours at 37 ° C. The excess biotin probe was removed by three changes in the SDS at 0.1% 2x SSPE for 10 minutes at 37 ° C followed by an incubation with TBST for 5 minutes. The biotin probe of the invention was visualized by performing the same protocol described in the direct spotting method.
Results Using this procedure it was found that the limit of detection of the biotin probe according to the invention was less than 10 pg.
Operation in In Situ Hybridization The test material consisted of 6 μm paraffin sections from an HPV-6 positive cervical condyloma mounted on glass plates coated with organosilane. The following protocol was applied (unless other steps were established at room temperature): • 1. Paraffin sections were dewaxed in 3 xylene changes and hydrated in graduated ethanol. 2. Cuttings were rinsed in TBST for 5 minutes. 3. The slices were digested in 0.25% pepsin in O.lN HCl for 30 minutes at 37 ° C, dehydrated in graduated methanol and air dried. 4. 10 μl of probe solution was applied to a cut and covered with a coverslip. The probe solution consisted of HPV-6 probe DNA with biotin, labeled according to the invention (batch # 061094) in a concentration of 2 ng / μl dissolved in hybridization mixture comprising 0.6 M NaCl, 0.06 sodium citrate. M, 35% formamide, 10% dextransulfate, 2.5x Denhardts and 10 μg / ml single-strand salmon sperm DNA. 5. The slices were placed on a hot plate at a fixed temperature of 95 ° C for 5 minutes to denature the probe and target DNA simultaneously. 6. Hybridization was carried out by placing the plates in a humid chamber at 37 ° C for 2 hours. 7. The coverslips were removed and the slides were washed in 3 changes of 15 M NaCl, 1.5 mM sodium citrate and 5% formamide for 10 minutes at 37 ° C. 8. The slides were rinsed in TBST. 9. Cuttings were incubated with Streptavidin AP conjugate (3DEA U / ml in TBST) for 15 minutes at 37 ° C. 10. The slides were washed in TBST (3x) and demineralized water (lx) for 5 minutes. 11. The sections were incubated with NBT / BCIP substrate solution for 15 minutes at 37 ° C. 12. The slides were washed in demineralized water (3x) for 1 minute and the slices were mounted in glycerol / gelatin.
Results Using the sections shown, the blue / purple precipitates at the sites of cells infected with HPV-6 and the smaller background in the remaining tissue.
Conclusions The results show that the DNA labeled according to the invention has good limits of detection. The method hereof is very suitable for research, for the routine use and for the industrial production of labeled nucleic acids, since the method is quick and easy to carry out, very sensitive, and does not include any enzymatic step, the which is highly reproducible and adjusts to a low total production cost. The method of the invention offers a useful alternative that matches conventional non-isotopic labeling methods.
General references 1. Maniatis * T., Sambrook J., Fritsch E. F., Molecular Cloning, Second Edition, Cold Spring Harbor
Laboratory Press, ISBN 0-8769-309-6. 2. Keller G. H., Manak M.M., DNA probes, Stockton Press, ISBN 0-333-47659-X.
Applications 1. The use of Pt-DNA linkers of the invention in the so-called LIDIA technique: Immunoassay Linked to DNA. The LIDIA technique allows the quantitative analysis of small amounts of DNA (or RNA), for example, after PCR amplification of the starting material. The technique is sensitive and specific, due to the use of DNA probes (specific RNAs) according to the invention, and easy to carry out, due to the Pt labeling steps of the rapid DNA (RNA) of the invention.
Description of the technique: The technique uses fast Pt labeling compounds of the invention to label DNA (RNA) probes. This technique is possible with 3 different methods. 1. Linking DNA probe molecules to a surface by using a Pt compound according to the invention, which crosslinks the DNA molecules irreversibly with "plastic, nylon or nitrocellulose." Detection of DNA targets it can then be done using 7DNA / RNA probes labeled in the classical form (trademark or chemical modification, random priming) 2. Link a detectable group to DNA, convert a DNA molecule into a so-called DNA probe. of DNA to a surface can then be carried out using the classical techniques known in science
(covalent binding to specially treated microtiter plates, support of DNA molecules on nitrocellulose or binding of DNA molecules to nylon membranes.) 3. A combination of techniques 1 and 2.
Method 1 An immobilized -DNA probe can be used to capture specific target molecules in a sample, using a hybridization technique. Detection of formed hybrids can be effected using different techniques, for example, a second labeled DNA probe can be used to hybridize to a different site on the target DNA molecule to form a sandwich hybrid. The mark can then be detected using the state of immunological detection techniques and tinting or staining techniques.
Method 2 A volume containing detectable (amplified) DNA (RNA) is directly labeled according to the protocol according to the invention. The excess mark is removed by adding NaDDTC or Thiourea. This method distinguishes itself from the other techniques by the fact that the target molecule is labeled in contrast to other assays, where the labeled DNA (RNA) probes are used to detect the target. The rapid binding capacity of the Pt-labeled compound of the invention allows a DNA binding step as a routine step in a diagnostic test procedure (normal binding times are 60 minutes at 65 ° C). A second step is carried out in a pre-coated microtiter plate with a specific target probe. It is allowed to incubate until the formation of a marked "stable target" and the probe is hybridized. Direct labeling of target molecules allows the omission of laborious double hybridization techniques, where a probe is used to trap the target and another probe is used to detect the immobilized target. In this method, the probes are conveniently linked to the microtitre plate to the surface of the wells. The second incubation step has the character of a liquid hybridization and therefore, can be carried out very quickly. This is one of the main innovative features of this method of quantitative DNA hybridization techniques.
Method 3 For both the immobilization of the DNA probes or DNA targets and for the labeling of the DNA probes and targets, the newly developed Pt system can be used. These two DNA binding techniques can be combined in one assay, where both the "sensor" and the "detector" are linked to a second substance (either a detectable group, such as biotin, digoxigenin or similar carrier surface). a plastic bar, microtitre plate or a membrane).
Examples of the technique: the detection of microorganisms related to STD in human diagnoses (Chlamydia, Syphilis, AIDS, Herpes, Gonorrhea, Hepatitis B).
2. The use of the Pt-DNA labels of the invention in combination with the procedures and formats of test strips. Rod of ADN'X The technique of the rod of DNA allows' in qualitative and semiquantitative analysis of small amounts of DNA
(or RNA), for example, after a PCR amplification or freely present in body fluid samples
(blood, urine, sweat, etc.). The technique is sensitive and specific, due to the use of specific DNA (RNA) probes, and easy to carry out due to the Pt labeling steps of the rapid DNA (RNA) according to the invention. The universal marking characteristics of the newly developed Pt mark can be used in 3 ways to achieve the union of a DNA (RNA) molecule. 1. It can be used to bind a detectable label group to a polynucleotide sequence. 2. It can be used to bind polynucleotide sequences irreversibly to a solid phase (plastic, membranes, latex beads, hydrosols or microtiter plate wells). 3. A combination of 1 and 2.
ad 1: In this example, there is a double method for the detection of biolithic biological analytes in test samples. First, a DNA probe can be labeled with the newly developed Pt labeling compound. This labeled probe can then be used to detect. pre-formed íbridos on a membrane formed between the target DNA sequence and a primary sequence. It is essential in this method, that the primary probe recognizes a different sequence on the target instead of the secondary probe r labeled with Pt. In practice, this can be achieved for example with the hybridization of RNA, where a probe is used. POLY A as a primary probe to immobilize all RNA (recognizable by its polyT tails) to a membrane .. The second method differs slightly, in that in this case, the target can be marked in the test sample fluid, due to the fast and very specific Pt marking characteristics. A procedure similar to this would comprise the capture of the target labeled with a specific unlabeled DNA probe immobilized on a suitable membrane. From here, a rod version for DNA / RNA applications.
ad 2: To immobilize DNA or target DNA probes, an unlabeled Pt compound (ie, a compound of P_t with 2 free binding sites) can be used to act as a bridge between the DNA and the surface of the carriers ( plastic, membrane, microtitre plates, etc.). This greatly increases the usefulness of DNA sequences as carrier molecules in diagnostic assays, since there are few known substances in science that readily bind to DNA in a spontaneous manner. Introducing this Pt bridge molecule to a broad field of applications for DNA technology has become achievable.
ad 3: a combination of examples 1 and 2 General: the use of the Pt compound of the invention in latex or hydrosol tests is particularly interesting. The compound 'allows the coupling of DNA molecules to small particles. DNA molecules can be hybridized with target material. A positive reaction is visualized by means of an "agglutination of the particles, due to the crosslinking of compounds of hybrid DNA particles." A test similar to this can be made quantitative, the speed of the agglutination can be graduated and measured at a wavelength. Especially, gold particles have the intrinsic characteristic that a deviation in the optimal wavelength occurs after agglutination.
3. Detection of DNA probes in Plate of the invention with the technique of amplification with silver. The DNA / RNA probe in plate can be used in the hybridization methods to detect DNA / RNA sequences in the sample material. The introduction of a platinum compound at the target site, allows the deposition of Ag molecules in a chemical reaction specially designed to reduce ionic silver to metallic silver. At the site of a Pt nucleus occurs a decomposition of metallic silver (black) due to the catalytic effect of the Pt nucleus. The ionic silver is reduced by means of a reducing agent (for example, Na borohydride, Hydroquinone) in solution. In a constant ratio, the amount of silver deposited on the Pt is proportional to the incubation period. Visualization of a non-visible Pt nucleus can be achieved by empirically observing the appearance of a black spot on the test sample. The black spots indicate the binding site of the specific probes and thus, the site of specific site location. The technique allows a quick and easy diagnostic procedure for the detection of various microorganisms and translocations / genetic abnormalities. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of "the invention.
Claims (22)
- CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1 . A method for labeling nucleotides, characterized in that it comprises the steps of: reacting a reactive portion of a linker of the formula wherein X represents any stabilizing bridge, and wherein A and B represent the same or different reactive portions, with an electron donor portion of a separator, which separator comprises a chain having at least four atoms and at least one hetero atom in the chain, separator which further comprises the electron donor portion at one end of the chain and a reactive portion at the other end of the chain; reacting the reactive portion of the separator with a mark; reacting the other reactive portion of the binder with a nucleotide.
- 2. The method according to claim 1, characterized in that the label is biotin, avidin, streptavidin, digoxigenin or a functional equivalent thereof,
- 3. The method according to claim 1 or 2, characterized in that the nucleotide is adenine. , thymidine, cytosine, guanine or uridine or a derivative thereof.
- 4. The method according to any of the preceding claims, characterized in that the donor portion of electrons is an amine or a thiolate anion.
- 5. The method according to claim 4, characterized in that the amine is an aromatic amine.
- 6. The method according to any of the preceding claims, characterized in that at least one hetero atom is an oxygen atom.
- The method according to any of the preceding claims, characterized in that the separator comprises no more than 20 carbon atoms in the chain, and wherein it is preferably an essentially unbranched chain.
- 8. The method according to claim 7, characterized in that the separator is 1,8-diamino-3,6-dioxaoctane.
- 9. The method according to claim 7, characterized in that the separator is an oligolysin or a polylysine,
- 10. The method according to claims 1-4, characterized in that the binding portion is reacted with a marker portion comprising a mark and a separator, marker portion which has the formula or the formula Z NH2 - (CH2) -NH2 \ / Pt marks g ' wherein X represents any stabilizing bridge, Z and Z 'represent a non-salient ligand and n is an integer from 2 to 10.
- 11. The method according to claim 10, characterized in that Z and / or Zr represent a group NH3, NH2R, NHR2 or NR3, in doh of R represents an alkyl group having from 1 to 6 carbon atoms.
- The method according to any of the preceding claims, characterized in that X represents an aliphatic diamine.
- 13. The method according to the claim 12, characterized in that X represents an aliphatic diamine having 2-6 carbon atoms.
- 14. The method according to the claim 13, characterized in that X is an ethylene diamine group.
- 15. The method according to claims 12-14, characterized in that one or both of the nitrogen atoms of the aliphatic diamine are protected.
- 16. The method of compliance with the claim 15, characterized in that one or both of the nitrogen atoms of the aliphatic diamine "*" are substituted with an alkyl group of 1 to 6 carbon atoms
- 17. The method according to the claim 16, characterized in that one or both of the nitrogen atoms of the aliphatic diamine are substituted with one or two methyl groups.
- 18. The method according to any of the preceding claims, characterized in that A and / or B are selected from the group consisting of N03 ~, S03", Cl ~, I", or other halogens.
- 19. The method according to any of the preceding claims, characterized in that A and B are the same.
- 20. A labeled nucleotide characterized in that it can be obtained by a method of compliance any of the preceding claims.
- 21. The labeled nucleotide according to claim 20, characterized in that the separator is an oligolysin or a polylysine.
- 22. A marker substance, characterized in that it has the formula marker wherein X represents a stabilizing bridge, A represents a reactive portion and the marker portion comprises a label and a separator, which comprises a chain having at least four atoms and at least one heteroatom in the chain, which separator comprises in addition the electron donor portion at one end of the distal separator of the mark.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96202792.6 | 1996-10-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA99003239A true MXPA99003239A (en) | 2000-09-04 |
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