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Photochemical Method of Labelling Nucleic Acids for Detection in Hybridization Assays

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Photochemical Method of Labelling Nucleic Acids for Detection in Hybridization Assays Europaisches Patentamt 3 European Patent Office © Publication number: 0 187 332 Office europeen des brevets A2 © EUROPEAN PATENT APPLICATION © Application number: 85116199.2 © IntCI.4: C 12 Q 1/68 //G01N33/532 © Date of filing: 18.12.85 © Priority: 10.01.85 US 690336 © Applicant: Molecular Diagnostics, Inc. 400 Morgan Lane West Haven, CT.06516(US) © Date of publication of application: 16.07.86 Bulletin 86/29 © Inventor: Dattagupta, Nanibhushan 470 Prospect Street © Designated Contracting States: New Haven, CT 0651 1 (US) AT BE CH DE FR GB IT LI LU NL SE © Representative: Jesse, Rarf-ROdiger, Dr. et al, Bayer AG Konzernverwaftung RP Patentabteilung D-5090 Leverkusen 1 Bayerwerk(DE) © Photochemical method of labelling nucleic acids for detection in hybridization assays. © In a test for the presence of a particular nucleic acid in a test sample wherein the same is subjected to hybridizing conditions with a Ibeled nucleic acid probe, and the product is assayed for the presence of said label, the improvement wherein said probe comprises a probe according to claim 1. A labeled nucleic acid probe comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemical- ly linked to the nucleic acid component, (c) a label and (d) a spacer chemically linking (b) and (c). Croydon Printing Company Ltd This application relates to the provision of labeled nucleic acid labeled probes suitable for hybridization assays. In European Patent Application No. 84107624.3 there is described a photochemical method of preparing labelled nucleic acids. The assays are of the type described in in European Application No. 841 07248.1. The labelled probes of EP 84107624.3 com- prise (a) a nucleic acid component, (b) a nucleic acid- binding lignad photochemically linked to the nucleic acid component, and (c) a label chemically linked to (b). Those probes generally perform quite satisfactorily but in some instances it is desirable to improve their performance, for example, it is an object of the present invention to provide a probe of improved solubility, to increase the efficiency of preparing such probes, and to improve the sensitivity of the probe in assay. These and other objects and advantages are realized in accordance with the present invention pursuant to which there is provided a labeled nucleic acid probe comprising (a) a nucleic acid component (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, (c) a label and (d) a spacer chemically linking (b) and (c). Advantageously the spacer includes a chain of up to about 50 atoms, preferably about 2 to 20 atoms, selected from the group consisting of carbon, oxygen, nitrogen and sulfur. Such spacer may be the polyfunctional of a member selected from the group consisting of peptide, hydrocarbon, polyalcohol, polyether, polyamide, polyimine and carbo- hydrate, e.g. -glycyl-glycyl-glycyl- or other oligo- peptide, an -amino-alkane-carbonyl radical such as -NH (CH2y5-CO-, a spermine or spermidine radical, an w-alkanediamine radical such as -NH-(CH2)6-NH or -HN-CH2-CH2-NH, or the like. Sugar, polyethylene oxide radicals, glyceryl, pentaerythritol, and like radicals can also serve as the spacers. These spacers can be di- rectly linked to the nucleic acid-binding lignad and/or the label or the linkages may include a divalent radi- cal of a coupler such as dithiobis succinimidyl propionate, 1,4-butanediol diglycidyl ether, a diisocyanate, carbodiimide, glyoxal, glutaraldehyde or the like. The spacer can be incorporated at any stage of the process of making the probe defined hereinabove. Thus the sequence can be any of the following: etc. The conditions for the individual steps are well known in chemistry. As described in EP 84107624.3, the nucleic acid is joined to the.ligand photo- chemically, employing a photoreactive nucleic as a furo- coumarin or a phenanthridine compound or a non-inter- calator compound such as netropsin, distamycin, Hoechst 33258 and bis-benzimidazole to link the nucleic acid to a label which can be "read" or assayed in conventional manner, including fluorescence detection. The end product is thus a labeled nucleic acid probe comprising (a) a nucleic acid component, (b) an intercalator or other nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to (B). Angelicin derivatives have the following formula Many other compounds with different R's can be synthesised following published procedures. = H and = Example with R1 and R3 methyl R2 R4 -CH2NH2. (RNH2). with X = NH2 has been synthesised alkoxyl, carbonyl, R1 - R4 can be -H, -OH, alkyl, carbalcoxyl R'1 and R'2 can be -H, -OH, alkyl, alkoxyl, carboxyl, carbalkoxyl and X-Y where X is -C-(CH2)n-Y -N-(CH2)n-Y -C-N-(CH2)n-Y -C-N-polyamine-Y degree of polyamine = 1-30 -C-N-polyether-Y -C-N-polyalcohol-Y -C-S- -C-O- Y is a label as biotin. Reaction of 3 with R*NH2 where R*NH2 is or or Angelicin s as in P 11 Amt as in P 12 Daunomycin as in P 13 Amino acridines as in P (14) Any other intercalator described can be modified simi- larly to carry a linker and a label for nucleic acids. Anthracyclines: photochemical reactions of anthracyclines are known. Many other analogues have been synthesised and found naturally. The novel photochemical method provides more favorable reaction conditions than the usual chemical coupling method for biochemically sensitive substances. By using proper wavelengths for irradiation, DNA, RNA and proteins can be modified without affecting the native structure of the polymers. The nucleic acid-binding ligand, hereinafter exemplified by an intercalator, and label can first be coupled and then protoreacted with the nucleic acid or the nucleic acid can first be photo- reaction with the intercalator and then coupled to the label. A general scheme for coupling a nucleic acid, exemplified by double-stranded DNA, to a label such as a hapten or enzyme is as follows: Where the hybridizable portion of the probe is in a double stranded form, such portion is then denaturated to yield a hybridizable single stranded portion. Al- ternatively, where the labeled DNA comprises the hybri- dizable portion already in single stranded form, such denaturization can be avoided if desired. Alternatively, double stranded DNA can be labeled by the approach of the present invention after hybridization has occurred using a hybridization format which generates double stranded DNA only in the presence of the sequence to be detected. To produce specific and efficient photochemical products, it is desirable that the nucleic acid component and the photoreactive intercalator compound be allowed to react in the dark in a specific manner. For coupling to DNA, aminomethyl psoralen, aminomethyl angelicin and amino alkyl ethidium or methi- dium azides are particularly useful compounds. They bind to double-stranded DNA and only the complex produces photoadduct. In the case where labeled double-stranded DNA must be denaturated in order to yield a hybridizable single stranded region, conditions are employed so that simultaneous interaction of two strands of DNA with a single photoadduct is prevented. It is necessary that the frequency of modification along a hybridizable single stranded portion of the probe not be so great as to substantially prevent hybridization, and accordingly there preferably will be not more than one site of modi- fication per 25, more usually 50, and preferably 100, nucleotide bases. Angelicin derivatives are superior to psoralen compounds for monoadduct formation. If a single- stranded probe is covalently attached to some extra double- stranded DNA, use of phenanthridum and psoralen compounds is desirable since these compounds interact specifically to double-stranded DNA in the dark. The chemistry for the synthesis of the coupled reagents to modify nucleic acids for labelling, described more fully hereinbelow, is similar for all cases. The nucleic acid component can be singly or doubly stranded DNA or RNA or fragments there of such as are produced by restriction enzymes or even relatively short oligomers. The nucleic acid-binding ligands of the present invention used to link the nucleic acid component to the label can be any suitable photoreactive form of known nucleic acid-binding ligands. Particularly preferred nuc- leic acid-binding ligands are intercalator compounds such as the furocoumarins, e.g., angelicin (isopsoralen) or psoralen or derivatives thereof which photochemically will react with nucleic acids, e.g., 4'-aminomethyl-4,5'- dimethyl angelicin, 4'-aminomethyl-trioxsalen (4'-aminomethyl-4,5', 8-trimethyl-psoralen, 3-carboxy-5- or -8-amino- or -hydroxy-psoralen, as well as mono- or bis-azido aminoalkyl methidium or ethidium compounds. Photoreactive forms of a variety of other intercalating agents can also be used as exemplified in the following table: Particularly useful photoreactive forms of such intercalating agents are the azidointercalators. Their reactive nitrenes are readily generated at long wavelength ultraviolet or visible light and the nitrenes of arylazides prefer insertion reactions over their rearrangement products fsee White et al, Methods in Enzymol. 46:644(1977)]. Representative azidointercalators are 3-azidoacridine, 9-azidoacridine, ethidium monoazide, ethidium diazide, ethidium dimer azide [Mitchell et al, JACS 104:4265(1982)], 4-azido-7-chloroquinoline, and 2-azidofluorene. Other useful photoreactable intercalators are the furocoumarins which form [2+2] cycloadducts with pyrimidine residues. Alkylating agents can also be used such as bis- chloroethylamines and epoxides or aziridines, e.g., aflatoxins, polycyclic hydrocarbon epoxides, mitomycin, and norphillin A. The label which is linked to the nucleic acid component according to the present invention can be any chemical croup or residue having a detectable physical or chemical property. The label will hear a functional chemical group to enable it to be chemically linked to the intercalator compound. Such labeling materials have been well developed in the field of immunoassays and in general most any label useful in such methods can be applidd to the present invention.
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