CA1222705A - Fast photochemical method of labelling nucleic acids for detection purposes in hybridization assays - Google Patents

Abstract

ABSTRACT

A labeled nucleic acid probe comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to the nucleic acid-binding ligand. The label can be a specifically bindable ligand such as a hapten or biotin, an enzyme such as a .beta.-galactosidase or horse radish peroxidase, a fluorescent radical, a phycobiliprotein, a luminescent radical, or a radioisotope. The probe can be used in assays of nucleic acids, taking advantage of the ability of the nucleic acid component to hybridize.

Description

~22;27~5 The present invention relates to a photochemieal method of labelling nucleic acids for detection purposes in hybridization assays for the determination of specifie polynueleotide sequenees.
The most effieient and sensitive method of detection of nucleic acids such as DNA after hybridization requires radioactively labelled DNA. The use of autoradiography and enzymes makes the assay time eonsuming and requires experienced teehnical people. Recently, a non-radioactive method of labelling DNA has been described using the method of niek translation to introduce biotinylated U
residue to DMA replaeing T. The biotin residue is then assayed with antibiotin antibody or an avidin eontaining system. The deteetion in this ease is quicker than autoradiography but the method of niek translation is a highly skilled art. Moreover, biotinylation using biotinylated TUP works only for thymine-eontaining polynueleotides. Use of other nueleotide triphosphates is very diffieult beeause the ehemieal derivatization of A or G or C (eontaining -NH2) with biotin requires elaborate and highly skilled organie ehemists.
It is aeeordingly an objeet of the present invention to provide a simplified system for deteetion of nueleie aeids by hybridization assays, whieh system ean be easily produced and used without the disadvantages noted hereinabove.
These and other objeets and advantages are realized in aceordanee with the present invention pursuant to whieh the nueleic aeid is labeled by means of photoehemistry, employing a photoreaetive nueleic acid-binding ligand, e.g., an intercalator compound such as a furoeoumarin or a phenanthridine compound or a non-intercalator eompound such as netropsin, distamycin, ~r~

~2~ S
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 comprisin~ (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).
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 photoreacted with the nucleic acid or the nucleic acid can first be photoreacted 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:

Label Photoreactive Intercalator Labeled Double-Stranded DNA
Photoreactive Intercalator .>
Photoreactive ~ Intercalator hv \ Chemically - Fun _tionalized DNA

~ ~ +Label ~' Labeled DNA
Where the hybridizable portion of the probe is in a double stranded form, such portion is then denatured to yield a hybridizable single stranded portion.
Alternatlvely, where the labeled DNA comprises the hybridiæable 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 methidium 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 denatured 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 modification 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 phenanthridium 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.

~L27~7~5i The nucleic acid component can be singly or doubly stranded DNA or RNA or fragments thereof 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 Eorm of known nucleic acid-binding ligands. Particularly preferred nucleic 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, 4l-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:

Intercalator Classes and Representative Compounds Literature References A. Acridine dyes Lerman, J. Mol. Biol.
3:18(1961); Bloomfield et al, "Physical Chemis~ry of Nucleic Acids", Chapter 7, pp.
429-476, Harper and Rowe, NY(1974) proflavin, acridine Miller et al, Bio-orange, quinacrine, polymers 19:2091(1980) acriflavine 30 B. Phenanthridines Bloomfield et al, supra Miller et al, supra ethidium coralyne Wilson et al, J. Med.
Chem. 19:1261(1976 ellipticine, ellipticine Festy et al, FEBS
cation and derivatives Letters 17:321(1971);

27~
Kohn et al, Cancer Res.
35:71(1976); LePecq et al, PNAS (USA)71:
5078(197~); Pelaprat et al, J. Med. Chem.
23:1330(1980) C. Phenazines Bloomfield et al, supra 5-methylphenazine cation D. Phenothiazines ibid chlopromazine E. Quinolines ibid chloroquine quinine F. Aflatoxin ibid G. Polycyclic hydrocarbons ibid and their oxirane derivatives 3,4-benzpyrene benzopyrene diol Yang et al, Biochem.
epoxide, l-pyrenyl- Biophys. Res. Comm.
oxirane 82:929(1978~
benzanthracene-5,6-oxide Amea et al, Science 176:~7(1972) H. Actinomycins Bloomfield et al, supra actinomycin D
I. Anthracyclinones ibid ~-rhodomycin A
daunamycin J. Thiaxanthenones ibid miracil D
K. Anthramycin ibid 30 L. Mitomycin Ogawa et al, Nucl.
Acids Res., Spec.
Publ. 3:79(1977);
Akhtar et al, Can. J.
Chem. 53:2891(2975) M. Platinium Complexes Lippard, Accts. Chem.
Res. 11:21111978) N. Polyintercalators echinomycin Waring et al, Nature 252:653(1974);

~L~2~7~
Wakelin, Biochem. J.
157:721(1976) quinomycin Lee et al, Biochem. J.
triostin 173:115(1978): Huang BBM928A et al, Biochem. 19:
tandem 5537(1980): Viswamitra et al, Nature 289:
817(1981) diacridines I.ePecq et al, PNAS
(USA)72:2915(1975):
Carrellakis et al, Biochim. Biophys.
Acta 418:277(1976);
Wakelin et al, Biochem 17:5057(1978); Wakelin et al, FEBS Lett.
104:261(1979); Capelle et al, Biochem. 18:3354 (1979); Wright et al, Biochem. 19:5825(1980);
Bernier et al, Biochem.
J~ 199:479 (1981); King et al, Biochem. 21:4982 (1982) ethidium dimer Gaugain et al, Biochem.

2 17:5078(1978); Kuhlman et al, Nucl. Acids Res.
5:2629(1978); Marlcovits et al, Anal. Biochem.
94:259(1979~: Dervan et al, JACS 100:1968(1978);
ibid 101:3664(1979).
ellipticene dimers Debarre et al, Compt.
and analogs Rend. Ser. D. 284:
81(1977); Pelaprat et al, J. Med. Chem.
23:1336(1980) heterodimers Cain et al, J. Med.
Chem. 21:658(1978);
Gaugain et al, Biochem.
17:5078(1978) trimers Hansen et al, JCS
Chem. Comm. 162(1983);
Atnell et al, JACS
105:2913(1983) O. Norphillin A Loun et al, JACS 104:
3213(1982) s P. Fluorenes and ~luorenones Bloom~ield et al, supra f]uorenodiamines Witkowski et al, Wiss. Beitr.-Martin-Luther-Univ. Halle Wittenbery, 11(1981) Q. Furocoumarlns angelicin Venema et al, M~G, Mol. Gen. Genet.
179;1 (1980) 4,5'-dimethylangelicin Vedaldi et al, Chem.-Biol. Interact. 36:
275(1981) psoralen Marciani et al, Z.
Natur~orsch B 27(2):
196(1972) 8-methoxypsoralen Belognzov et al, Mutat.
Res. 84:11(1981);
Scott et al, Photochem.
Photobiol. 34:63(1981) 5-aminomethyl~8- Hansen et al, Tet. Lett.
methoxypsoralen 22:1847(1981) 4,5,8-trimethylpsoralen Ben-Hur et al, Biochem. Biophys.
Acta 331:181(1973) 4'-aminomethyl-4,5,8- Issacs et al, Biochem.
trimethylpsoralen 16:1058(1977) xanthotoxin Hradecma et al, Acta Virol. (Engl. Ed.) 26:
305(1982) khellin Beaumont et al, Biochim. Biophys.
Acta 608:1829(1980) R. Benzodipyrones Murx et al, J. Het.
Chem. 12:417(1975);
Horter et al, Photo-chem. Photobiol. 20:
407(1974~
S~ Monostral Fast Blue Juarranz et al, Acta Histochem. 70:130 (1982) ~L2~Z~5 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 o~ arylazides prefer insertion reactions over their rearran~ement products [see 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. Al~ylating agents can also be used such as bis- chloroethylamines and epoxides or a~iridines, 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 group or residue having a detectable physical or chemical property. The label will bear 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 applied to the present invention. Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem.(1976)22:1243), enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Pat.
Nos. 4,230,797 and 4,238,565), and enzyme inhibitors (see U.S. Pat. No. 4,134,792; fluorescers (see Clin.
Chem.(1979)25:353) and chromophores including phycobiliproteins; luminescers such as chemiluminescers and bioluminescers (see Clin. Chem.(1979)25:512, and ibid, 1531); specifically bindable ligands; and residues comprising radioisotopes such as 3H, 35S, 32p, 2~

125I, and 14C. Such labels are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors). For example, a cofactor-labeled nucleic acid can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme. A hapten or ligand (e.g., biotin) labeled nucleic acid can be detected by adding an antibody or an antibody fragment to the hapten or a protein (e.g., avidin) which binds the ligand, tagged with a detectable molecule. Such detectable molecule can be some molecule with a measurable physical property (e.g., fluorescence or absorbance) or a participant in an enzyme reaction te.g., see above list). For example, one can use an enzyme which acts upon a substrate to generate a product with a measurable physical property. Examples of the latter include, but are not limited to, ~-galactosidase, alkaline phosphatase, papain, and peroxidase. For in situ hybridization studies, ideally the final product is water insoluble. Other labels will be evident to one of the ordinary skill in the art.
The label will be linked to the intercalator compound by direct chemical linkage such as involving covalent bonds, or by indirect linkage such as by the incorporation of the label in a microcapsule or liposome which in turn is linked to the intercalator compound. Methods by which the label is linked to the intercalator compound are essentially known in the art and any convenient mèthod can be used to perform the present invention.
Advantageously the intercalator compound is first combined with the label chemically and thereafter combined with the nucleic acid component. For example, since biotin carries a carboxyl group it can be combined with a furocoumarin by way of amide or ester ~2~7~
fo.rmcl-tion WithOllt interferillg with t:]le photochemical reactivlty oE the furocoumarirl OL the hiolocl-cal ac-tivity c~:~ tlle biotin, e.c~., (i) N ~ (CII2)~ ~ C - ~

Biotin-N-hydroxysuccirlimide or 11 -~ P~H2 ( ii ) 0~ ~ ~ ,. S o ~I/N ~ ~ (C~l2)4- C ~ ~ ~ ~N02 Biotln-p-nitrophenyl ester-0~ O

or carbodiimide Biotin + ROI-I . ~ Biotin CO OR
By way of example, 2 2 T-~

( C112 ) ~; COO--~/ \,~ M2 Amt ~-J
Biotin n;.trophenyl ester -5 ~ o ~2~7~5 ~NII
( ( ~ E E 2 ) ~ C ON 1-1 C 112 Other aminomethy]ange~ic:in, psoralen and phenanthridium derlvatives can be simi.larly reacted, as can phenanthri~ium halides and derivatives thereof such as amioopropyl methidium chloride, i.e.

H2N ~ ~ N~2 Cl O C N~l - CH2 - CH2 - CH2 N 2 _,,_,,, [see Hertzberg e-t al, J. Amer. Chem. Soc.
104:313(1982)]
Alternative].y a bifunctional reagent such as dithiobi.s succinimidyl propionate or 1,4--butanediol diglycidyl ether can be used directly to couple the photochemically reactive molecule with the label where the reactants have alkyl amino residues, again in a known manner with regard to solvents, proportions and reaction conditions. Certain bifunctional reagents, possibly glutaraldyde may not be suitable because, while they couple, they may modify the nucleic acid and -thus interfere with the assay. Routine precautions can be taken to prevent such aifficulties.
The particular sequence in making the labeled nucleic acid can be varied. Thus, for example, an amino-substituted psoralen can first be photometrically coupled with a nucleic acid, the product having pendant amino groups by which it can be coupl.ed to the label.

. _ _ . . . . ... , . ... . _ . . _ _ _ _ _ _ ~222~
Alternatively, the psoralen can first be coupled to a label such as an enzyme and then to the nuclelc acid.
The spacer chain length between the nucleic acid-binding ligand and the label can be extended via hydrocarbon or peptide. A typical example involves extending an 8-hydro~y psoralen derivative with an alkyl halide,according to the method described by J.
L. DeCout and J. Lhomme, Photochemistry Photobiology, 37, 155-161 (1933). The haloal]cylated derivative is then reac-ted either with thiol or amines to produce the reactive residue, as has been described by W. A.
Saffran et al., Proc. Natl. Acad. Sci., U.S.A., 79, 4594 (1982) If the label is an enzyme, for example, the product will ultimately be placed on a suitable medium and the extent of catalysis will be determined. Thus, if the enzyme is a phosphatase the medium could contain nitrophenyl phosphate and one would monitor the amount of nitrophenol generated by observing the color. If the enzyme is a ~galactosidase the medium can contain o-nitrophenyl-D-galacto-pyranoside which also will liberate nitrophenol.
The labeled nucleic acid of the present invention is applicable to all conventional hybridization assay formats, and in general to any format that is possible based on formation of a hybridization product or aggregate comprising the labeled nucleic acid. In particular, the unique labeled probe of the present invention can be used in solution and solid-phase hybridization formats, including, in the latter case, formats involving immobilization of either sample or probe nucleic acids and sandwich formats.
The labeled nucleic acid probe will comprise at least one single stranded base sequence substantially complementary to or homologous with the sequence to be detected. However, such base sequence need not be a single continuous polynucleotide segment, but can be 7q~5 comprised of two or more individual seqments interrupted by nonhomologous sequences. These nonhomologous se~uences can be linear or they can be sel~-complementary and form hairpin loops. In addition, the homologous region o~ the probe can be flanked at the 3' - and 5' - terminii by nonhomologous sequences, such as those comprising the DNA or RNA of a vector into which the homologous sequence had been inserted for propagation. In either instance, the probe as presented as an analytical reagent will exhibit detectable hybridization at one or more points with sample nucleic acids of interest. Linear or circular single stranded polynucleotides can be used as the probe element, with major or minor portions being duplexed with a complementary polynucleotide strand or strands, provided that the critical homologous segment or segments are in single stranded form and available for hybridization with sample DNA or RN~. Useful probes include linear or circular probes wherein the homologous probe sequence is in essenially only single stranded form [see particularly, Hu and Messing, Gene 17:~71tl982)].
The labeled probe of the present invention can be used in any conventional hybridization technique. As improvements are made and as conceptually new formats are developed, such can be readily applied to the present labeled probe. Conventional hybridization formats which are particularly useful include those wherein the sample nucleic-ac-ids or the polynucleotide probe is immobilized on a solid support (solid-phase hybridization~ and those wherein the polynucleotide species are all in solution (solution hybridization).
In solid~phase hybridization formats, one of the polynucleotide species participating in hybridization is fixed in an appropriate manner in its single stranded form to a solid support. Useful solid supports are well known in the art and include those ~2~ S
which bind nucleic acids either covalently or non-covalently. Noncovalent supports which are generally understood to invo.lve hydrophobic bonding include naturally occurring and synthetic polymeric materials, such as nitrocellulose, derivatized nylon, and fluorinated polyhydrocarbons, in a variety of forms such AS flters or solid sheets. Covalent binding supports are also useful and comprise materials having chemically reactive groups or groups, such as dichlorotriazine, diazobenzyloxymethyl, and the like, which can be activated for binding to polynucleotides.
A typical solid-phase hybridization technique begins with imrnobilization of sample nucleic acids onto the support in single stranded form. This initial step essentia]ly prevents reannealing of complementary strands from the sample and can be used as a means for concentrating sample material on the support for enhanced detectability. The polynucleotide probe is then contacted with the support and hybridization detected by measurement of the label as described herein. The solid support provides a convenient means for separating labeled probe which has hybridized to the sequence to be detected from that which has not hybridized.
Another method of interest is the sandwich hybridization technique wherein one of two mutually exclusive fragments of the homologous sequence of the probe is immobilized and the other is labelled. The presence of the polynucleotide sequence of interest results in dual hybridization to the immobilized and labeled probe segments See Methods in Enzymology 65:468(1980) and Gene 21:77-85(1983~ for further details.
The invention will be further described in the following examples wherein parts are by weight unless otherwise expressed.

~27~)S
Example 1:
50 mg of N-hydroxysuccinimido biotin is dissolved in 2 ml dimethylsulfoxide tsoln A). 10 mg of 4' aminomethyl trioxsalen (structure 1) (or other aminoalkyl compounds) is dissolved in 10 ml (soln B) aqueous buffer (e.g., 10 mM sodium tetraborate, pH
adjusted with ~Cl) solution pH~ 8. Solution (~J and (BJ are mixed in a volume ratio of 1:10 and weight ratio of 10:1, so that the activated hapten is present in large excess. The reaction is allowed to proceed at 35C for 1 hour. The extent of the reaction is monitored by thin layer chromatography - on silica gel plates with a fluorescence indicators in a solvent 1/1/8 -- methanol/Acetic acid/chloroform. Under these TLC conditions unreacted aminomethyl trioxalane moves with the solvent front whereas the product has a slower mobility. Biotin does not show any fluorescence but the adduct fluoreces because of trioxsalen. Growth of the new fluorescent spot and disappearance of the original fluorescent spot indicates the extent of product formation. Since the activated biotin is in large excess, fluorescence corresponding to the starting material vanishes on TLC after the completion of reactionO Excess active biotin is reacted with glycyl-glycine or lysine. The presence of amino acid biotin product does not interfere with the photochemical reaction of psoralen-biotin compounds with DNA. Hence, a purification step after the above reaction is not essential.
Example 2:
100 mg of biotin nitrophenyl ester is dissolved in dry DMSO 12-5 ml) and 10 mg of 4'-aminomethyl trioxsalen is dissolved in dry DMSO (5 ml). The two solutions are mixed in a molar ratio so that biotin nitrophenyl es-ter is about ten times with respect to 4'-aminomethyl trioxsalen. 100 ml of triethylamine is added to the mixture and shaken well. The progress of reaction is checked by TLC and excess unreacted biotin nitrophenyl ester is reacted wlth lysine as in Example 1. The reaction is allowed to proceed for 1 hour at 35C and then lysine is added to quench the reaction.
After the reaction, DMSO is evaporated under vacuum and the gummy residue is taken in methanol and can be chromatographically purified on an LH 20 column, using methanol as an eluant. I'he last step is not essential for the photochemical interaction of psoralen adduct with DNA.

Example 3.
Biotin can be coupled to aminoalkyl hydroxyalkyl compounds by carbodiimide mediated reaction. lOmg biotin is dissolved in 1 ml dimethyl formamide. To the solution, 5 mg of 4'-hydroxymethyl trioxsalen is added followed by lOmg dicyclohexyl carbodiimide. The reaction is allowed to proceed for 20 hours at room temperature, dicyclohexylurea precipitate is removed and the product is recovered by removing DMF under vacuum. The same reaction can be performed in pyridine.
The foregoing examples will be give similar results if the animoalkyl trioxsalen is replaced by other aminoalkyl furocoumarins, phenanthridium halides, and the like.

Example 4 Coupling of an enzyme to a photoactive amino compound and then covalent attachment to DNA:
A typical example is given with papain. 0.1 mg/ml of papain solution in 100 mM phosphate buffer (pH 8) is added to 10 mg/ml of amino methyl trioxsalen. The final solution should be 1:1 with respect to volume of enzyme and photoactivator solution. Then solid dithiobis-succinimidyl propionate or dimethyl s suberimidate ls added to a final concentrakion of 20 ~g/ml. The pH is continuously monitored and maintained at the original value by 0.001 M sodium hydroxide.
After adding the crosslinker twice, the reaction is allowed to proceed for 30 minutes at room temperature.
The free photoactive amine is separated from the enzyme-bound compounds by gel filtratio~ on Sephadex G-lO~ The adduct is excluded along with the free protein and protein-protein conjugates. Most of these impurities have very little effect on DNA binding. Any enzyme which has been modified and still retains its activity can be coupled similarly.
After the purification, the enzyme conjugate is mixed with DNA in a~ueous buffer (pH 7.5) and irradiated at 390 nm for 1 hour. The adduct is separated from the unreacted residues on Sephadex (G-100) column. The activity is tested as follows:
DNA-enzyme conjugate is dialyzed against lO mM EDTA -containing buffer (pH 6~2). To 8 ml of the DNA-enzyme solution, 10 ml of 60 mM mercaptoethanol and 1 ml 50 m mol cysteine (freshly prepared) are added. This is treated as enzyme solution. The substrate solution is prepared as follows:

592 mg benzoyl-L-arginine ethyl ester hydrochloride is dissolved in 30 ml water (BAEE).
To this BAEE solution, 1.6 ml 0.01 M EDTA, 1.6 ml 0.05 M cysteine, freshly prepared are added, pH is adjusted at 6.2 and the final volume is made up to 42 ml.

Procedure Using a pH meter, the following test system has heen set up at 25C:
5 ml substrate 5 ml H2O

2~
5 ml 3 M NaCl 1 ml enzyme dilution The amount of 0.01 M NaOH in ml re~uired to maintain a pH of 6.2 is recorded. A five-minute period is generally satisfactory.

Since enzyme are not stable at higher temperatures, if the conjugates are used for hybridization assays, low temp~ratures should be used.
(Either oligonucleotides, or an ionic strength less than 2 m molar should be utilized so that hybridization can be effected at low temperature.) Exam~le 5-.

Identical products are generated if aminoalkylphotoactive compounds are photoreacted with DNA first, then with the proteins or enzymes or haptens. DNA (1 mg/ml) and amino methyl trioxsalen (0.1 mg/ml) are mixed in aqueous buffer pH(7.5), and photoirradiated at 390 nm for 1 hour; the product is precipitated with ethanol then redissolved in crosslinking buffer as in Example 4, and the rest of the procedure is similar.
If monoadduct formation is essential, monoazidoaminopropyl methidium or aminomethyl angelicin compounds are used under otherwise identical conditions.

Example 6:
A glycoprotein can be coupled by redox reaction to an aliphatic amine. A typical example is given below with horse radish peroxidase (HRPO) coupling to 4' aminomethyl trioxsalen. Identical conditions can be followed with any aminoalkyl compound.

~%7~5 Scheme:
P~riodate HRPO ~ (HRPO~-CHO
~I RNH2 Reduction (HRpo)cH2-NHR ~ (HRPO)-CH = N-R
NaBH4 ~E_riment:
10 mg HRPO (Sigma Chemical Co.) is dissolved in 2 ml freshly prepared 0.3 M sodium bicarbonate (pH 8.1).
To the enzyme solution, 200 microliter 1~
2,4-dini-trofluorobenzene in ethanol is added to block - and ~ -amino groups and some hydroxy groups of the enzyme. The mixture is gently shaken for one hour at room temperature. Then 2 ml 80 m molar sodium periodate in distilled water is added and mixed for 30 minutes at room temperature. In order to quench the unreacted periodate, ethylene glycol is added to a final concentration of 50 m Mol. The solution is dialyzed against 10 m molar sodium carbonate buffer (pH
9.5) in a cold room (~ 4~C). To the dialyzed solution, ~ 1 mg solid aminomethyl trioxsalen is added and the mixture is shaken gently for 1 hour at 25G. 10 mg sodium borohydride (NaB~4) solid is added and the reaction is allowed to proceed for 12 hours at 4C.
The adduct is dialyzed agains-t the DNA binding buffer and then photoreacted by mixing in 1:1 weight ratio (enzyme to DNA) as described before. The separation of the DNA-enzyme adduct from the enzyme is done by gel filtration on a Sephadex G-100 column where the adduct is excluded.
To improve the photochemical efficiency, blocking of reactive HRPO sites before oxidation with periodate may be done with allylisothiocyanate, as has been described by P.~. Nakane et al, Enzyme Labeled Antibodies for Light and Electron Microscopic ~2;~70S

Localizatlon of Antl~ens, J. ~istochem Cytochem, lb, 79Q (1966).
Unless stated otherw~se, all the react~on~ are performed in the darX or red llght ~ondltlons are maintained.
The peroxidase activity i5 measured by the followin~ method:
100-500 m~croliters of t~e sample are mlxed with 3 ml 14 mH para-Cresol in 50 m molar tris HCl buffer (pH 7.5). To thi~ 1 ml 1~ H202 is added. After 2 minutes, 3 ml 5 m molar sodium cyanides ;n water are added to quench the reaction. The fluorescence of the solution is measured at excitation 320 nm, emission 410 nm. H. Perschke and E. Broda, Nature 190, 257 (1961); U. Roth, ~ethods of Biochemical Analysis, vol. 17, ed. D. Glick, Interscience Publisher, N.Y., 1969, P. 236.

_xample 7: Assay for the label after DWA-DWA hybridization:
An illustrative example with a single ~tag~ DNA-DNA hybridization is presented here. The procedure used in the case of two-state hybridization can also be followed (Canadian Application ~o. 454,942 flled July 25, 1985).
Plasmid pBR322* ~New England Biolab~ ~s digested wlth the restriction endonuclease, Pst 1 and Pvu 1. This double algestlon produces one frasment of 126 base pair long D~A containing the part of ampicillln reslstance ~ene and another fragment of 423S base pair lon~ DNA. The 126 bp long fraBment is isolated hy running the double dl~est on 5~ polyscrylamide gel. A part of this DNA is labeled either wit~ biotin or with enzymes as described before and used as the labeled probe. For hybridlzation, Pst 1 cut pBR322 (for control) or the test sample DNA ls covalently linked to cellulose by photochemical method (Appllcatlon Serial ~o. 511,064, filed July 5, 1983 now U.S. patent no.
4,542,102 issued September 17, 1985), by cyanogen bromide *Trade ~ark - 21 -" -activation or by diazotization method (H. B~nemann, Nuclelc Acids Res., 10, 7181 (1982)).
The cel]ulose containing the denatured D~A is suspended in 5 m molar salt solution for hybridization with enzyme-coupled DNA or suspended in 2.4 M tetra-ethylammonium chloride when biotinylated DNA is used.
Then hybridization is done as described by ~. B~nemann in Nucleic Acids Research, 10, 7181 (1982) for the detection of the ampicillin resistance gene usiny 126 base pairs labeled fragment as the probe. In low salt, hybridization is done at 30-40C; in 2.4 M (high salt), it is done between 40 and 50C.
AEter hybridization, FITC-labelled avidine is used to assay for biotin or proper enzyme assay is done with the particles.
It will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

Claims (52)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE
IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A hybridizable labeled nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to (b).

2. A hybridizable labeled nucleic acid according to claim 1, wherein the nucleic acid-binding ligand is an intercalator compound selected from the group consisting of acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.

3. A hybridizable labeled nucleic acid according to claim 2, wherein the intercalator compound is a furocoumarin or a phenanthridine.

4. A hybridizable labeled nucleic acid according to claim 1, wherein the label (c) is a specifically bindable ligand.

5. A hybridizable labeled nucleic acid according to claim 4, wherein the label is a hapten.

6. A hybridizable labeled nucleic acid according to claim 4, wherein the label is biotin.

7. A hybridizable labeled nucleic acid according to claim 1, wherein the label (c) is an enzyme.

8. A hybridizable labeled nucleic acid according to claim 1, wherein the label (c) is a fluorescent or luminescent radical.

9. A hybridizable labeled nucleic acid according to claim 8, wherein the radical is from fluorescein.

10. A hybridizable labeled nucleic acid according to claim 1, wherein the label (c) is a phycobiliprotein.

11. A hybridizable labeled nucleic acid according to claim 6, wherein the chemical link between the biotin and (b) is effected via succinimidyl activation.

12. A hybridizable labeled nucleic acid according to claim 6, wherein (b) is the radical of an amino-substituted angelicin or psoralen and is linked to the biotin through an amide group.

13. A hybridizable labeled nucleic acid according to claim 7, wherein the enzyme is .beta.-galactosidase.

14. A hybridizable labeled nucleic acid according to claim 7, wherein the enzyme is horse radish peroxidase.

15. A hybridizable labeled nucleic acid according to claim 7, wherein the enzyme is papain.

16. A hybridizable labeled nucleic acid according to claim 1, wherein the nucleic acid component is in single stranded form.

17. A hybridizable labeled nucleic acid according to claim 1, wherein the nucleic acid component is in a double stranded form.

18. An adduct suitable for photochemical attachment to a nucleic acid probe, comprising a nucleic acid-binding ligand and a label chemically linked thereto.

19. An adduct according to claim 18, wherein the nucleic acid-binding ligand is an intercalator compound selected from the group consisting of acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.

20. An adduct according to claim 19, wherein the intercalator compound is a furocoumarin or a phenanthridine.

21. An adduct according to claim 18, wherein the label is a specifically bindable ligand.

22. An adduct according to claim 21, wherein the label is hapten.

23. An adduct according to claim 21, wherein the label is biotin.

24. An adduct according to claim 18, wherein the label is an enzyme.

25. An adduct according to claim 18, wherein the label is a fluorescent or luminescent radical.

26. An adduct according to claim 25, wherein the radical is from fluorescein.

27. An adduct according to claim 18, wherein the label is a phycobiliprotein.

28. An adduct according to claim 18, wherein the nucleic acid-binding ligand is an angelicin or psoralen carrying an amino substituent.

29. An adduct according to claim 24, wherein the enzyme is .beta.-galactosidase.

30. An adduct according to claim 24, wherein the enzyme is horse radish peroxidase.

31. A method of making a labeled hybridizable nucleic acid probe, which comprises contacting such nucleic acid with an adduct according to claim 18 and subjecting the nucleic acid and adduct to photochemical irradiation.

32. A method of making a labeled hybridizable nucleic acid, which comprises contacting a nucleic acid with a chemically-functionalized intercalator compound which bears an active group selected from group consisting of amine, carboxyl and hydroxyl groups, subjecting the nucleic acid and the intercalator compound to photochemical irradiation to effect a covalent reaction to photochemically link the nucleic acid to the intercalator compound, and subjecting the resultant reaction product to further reaction to introduce a label in place of the chemically-functionalized moiety.

33. A hybridizable labeled nucleic acid according to claim 1, wherein the nucleic acid-binding ligand is an intercalator compound selected from the group consisting of scridine dyes, phenanthridines, phenazines, phenothiazines and quinolines.

34. A method for determining a particular polynucleotide sequence in a test sample, comprising the steps of:
(a) combining the test sample with a polynucleotide probe having a base sequence substantially complementary to the sequence to be determined, wherein a nucleic acid-binding ligand is photochemically linked to a sequence selected from the group consisting of the sample sequence and the probe sequence, the nucleic acid-binding ligand being chemically linked to a detectable label moiety, and (b) detecting the formation of hybrids between the sample sequence to be determined and the probe sequence by measuring said detectable label moiety.

35. A method of claim 34, wherein the unlabeled one of the sample sequence and the probe sequence is immobilized.

36. A method of claim 34, wherein said probe sequence is a first probe sequence and is labeled and an immobilized form of a second probe sequence is combined with the test sample, the first and second probe sequences being complementary to mutually exclusive portions of the sample sequence to be determined.

37. A method according to claim 34, wherein the nucleic acid-binding ligand is an intercalator compound selected from the group consisting of scridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.

38. A method according to claim 37, wherein the intercalator compound is a furocoumarin or a phenanthridine.

39. A method according to claim 34, wherein the label (c) is a specifically bindable ligand.

40. A method according to claim 39, wherein the label is a hapten.

41. A method according to claim 39, wherein the label is biotin.

42. A method according to claim 34, wherein the label (c) is an enzyme.

43. A method according to claim 34, wherein the label (c) is a fluorescent or luminescent radical.

44. A method according to claim 43, wherein the radical is from fluorescein.

45. A method according to claim 34, wherein the label (c) is a phycobili-protein.

46. The method of claim 34, wherein the unlabeled sequence selected from the group consisting of the sample sequence and the probe sequence is immobilized.

47. The method of claim 34, wherein said probe sequence is a first probe sequence and is labeled and an immobilized form of a second probe sequence is combined with the test sample, the first and second probe sequences being complementary to mutually exclusive portions of the sample sequence to be determined.

48. A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) an intercalator compound photochemically linked to the nucleic acid component, and (c) a label chemically linked to (b), the labeled hybridizable nucleic acid produced by a process selected from the group consisting of (i) photochemically linking the nucleic acid component to the intercalator compound and chemically linking a label to the chemically linked intercalator compound and (ii) chemically linking a label to the intercalator compound to form a composite and photochemically linking said composite to the nucleic acid component.

49. A labeled hybridizable nucleic acid probe comprising (a) a nucleic acid component, (b) a nucleic acid binding ligand, (c) a photochemical linker coupling the nucleic acid component and the nucleic acid binding ligand and (d) a label chemically linked to the nucleic acid binding ligand.

50. A labeled hybridizable nucleic acid according to claim 49, wherein the photochemical linker is selected from the group consisting of dithiobis succinimidyl propionate and 1,4-butanediol diglycidyl ether.

51. An adduct suitable for photochemical attachment to a nucleic acid comprising (a) a nucleic acid binding ligand, (b) a photochemical linker coupling the nucleic acid component and the nucleic acid binding ligand and (c) a label chemically linked to the nucleic acid binding ligand.

52. An adduct according to claim 51, wherein the photochemical linker is selected from the group consisting of dithiobis succinimidyl propionate and 1,4-butanediol diglycidyl ether.