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Claims  |
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What is claimed is:
1. A labeled hydribizable nucleic acid comprising (a) a nucleic acid
component, (b) a nucleic acid-binding ligand photochemically linked to the
nucleic acid component thereby modifying the nucleic acid such that the
frequency of modification along a hybridizable single stranded portion of
the nucleic acid not be so great as to substantially prevent
hybridization, and (c) a label chemically linked to (b), said label
selected from the group consisting of fluorescein and phycobiliprotein.
2. A hydridizable 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 is fluorescein.
5. A hybridizable labeled nucleic acid according to claim 1, wherein the
label is a phycobiliprotein.
6. A hybridizable labeled nucleic acid according to claim 1, wherein (b) is
a radical of an amino-substituted angelicin or psoralen.
7. A hybridizable labeled nucleic acid according to claim 1, wherein the
nucleic acid component is in single stranded form.
8. An adduct suitable for photochemical attachment to a hybridizable
nucleic acid probe, comprising a nucleic acid-binding ligand and a label
chemically linked thereto, whereby linkage of the adduct to the nucleic
acid modifies the nucleic acid such that the frequency of modification
along a hybridizable single stranded portion of the nucleic acid not be so
great as to substantially prevent hybridization, wherein the adduct is
capable of being photochemically linked to the nucleic acid and wherein
the label is selected from the group consisting of fluorescein and
phycoiliprotein.
9. An adduct according to claim 8, wherein the nucleic acid-binding ligand
is an intercalator compound selected from the group consisting of acridine
dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and
quinolines.
10. An adduct according to claim 9, wherein the intercalator compound is a
furocoumarin or a phenanthridine.
11. An adduct according to claim 8, wherein the label is fluorescein.
12. An adduct according to claim 8, wherein the label is a
phycobiliprotein.
13. An adduct according to claim 8, wherein the nucleic acid-binding ligand
is an angelicin or psoralen carrying an amino substituent.
14. A method for determining a particular polynucleotide sequence in a test
sample, comprising the step of:
(a) combining the test sample with a polynucleotide probe having a base
sequence substantially complementary to the sequence to be determined,
wherein a mono-adduct forming 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, wherein the
label is selected from the group consisting of fluorescein and
phycobiliprotein, and
(b) detecting the formation of hybrids between the sample sequence to be
determined and the probe sequence by measuring said detectable label
moiety.
15. A method according to claim 14, wherein the nucleic acid-binding ligand
is an intercalator compound selected from the group consisting of acridine
dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and
quinolines.
16. A method according to claim 15, wherein the intercalator compound is a
furocoumarin or a phenanthridine.
17. A method according to claim 14, wherein the label is fluorescein.
18. A method according to claim 14, 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.
19. A labeled hybridizale nucleic acid according to claim 11, wherein the
linker group is selected from the group consisting of dithiobis
succinimidyl propionate and 1,4-butanediol diglycidyl ether.
20. An adduct suitable for photochemical attachment to a nucleic acid
comprising
(a) a nucleic acid-binding ligand,
(b) a linker group coupled to the nucleic acid-binding ligand and
(c) a label chemically linked through the linker group to the nucleic
acid-binding ligand, whereby linkage of the adduct to the nucleic acid
modifies the nucleic acid such that the frequency of modification along a
hybridizable single stranded portion of the nucleic acid not be so great
as to substantially prevent hybridization, wherein the adduct is capable
of being photochemically linked to the nucleic acid and wherein the label
is selected from the group consisting of fluorescein and phycobiliprotein.
21. An adduct according to claim 20, wherein the linker group is selected
from the group consisting of dithiobis succinimidyl propionate and
1,4-butanediol diglycidyl ether.
22. A hybridizable labeled nucleic acid comprising (a) a nucleic acid
component, (b) a nucleic acid-binding ligand photochemically linked to the
nucleic acid component thereby modifying the nucleic acid such that the
frequency of modification along a hybridizable single stranded portion of
the nucleic acid not be so great as to substantially prevent
hydridization, and (c) a label chemically linked to (b), wherein the
nucleic acid is modified at not more than one site per 50 nucleotide
bases.
23. A hybridizable labeled nucleic acid comprising (a) a nucleic acid
component, (b) a nucleic acid-binding ligand photochemically linked to the
nucleic acid component thereby modifying the nucleic acid such that the
frequency of modification along a hybridizable single stranded portion of
the nucleic acid not be so great as to substantially prevent
hybridization, and (c) a label chemically linked to (b), wherein the
nucleic acid is modified at not more than one site per 100 nucleotide
bases.
24. A method of making a labeled nucleic acid probe, which comprises
contacting such probe with an adduct according to claim 8 and subjecting
the probe and adduct to photochemical irradiation, wherein the nucleic
acid is modified at not more than one site per 50 nucleotide bases.
25. A method of making a labeled nucleic acid probe, which comprises
contacting such probe with an adduct according to claim 8 and subjected
the probe and adduct to photochemical irradiation, wherein the nucleic
acid is modified at not more than one site per 100 nucleotide bases.
26. A labeled hybridizable nucleic acid comprising
(a) a nucleic acid component,
(b) a nucleic acid-binding ligand photochemically linked to the nucleic
acid component thereby modifying the nucleic acid such that the frequency
of modification along a hybridizable single stranded portion of the
nucleic acid not to be so great as to substantially prevent hybridization,
(c) a linker group coupled to the nucleic acid-binding ligand and
(d) a label chemically linked through the linker group to the nucleic
acid-binding ligand, wherein the nucleic acid is modified at not more than
one site per 50 nucleotide bases.
27. A labeled hybridizable nucleic acid comprising
(a) a nucleic acid component,
(b) a nucleic acid-binding ligand photochemically linked to the nucleic
acid component thereby modifying the nucleic acid such that the frequency
of modification along a hybridizable single stranded portion of the
nucleic acid not to be so great as to substantially prevent hybridization,
(c) a linker group coupled to the nucleic acid-binding ligand and
(d) a label chemically linked through the linker group to the nucleic acid
binding ligand, wherein the nucleic acid is modified at not more than one
site per 100 nucleotide bases.
28. A labeled hybridizable nucleic acid comprising
(a) a nucleic acid component,
(b) an intercalator compound photochemically linked to the nucleic acid
component thereby modifying the nucleic acid such that the frequency of
modification along a hybridizable single stranded portion of the nucleic
acid not be so great as to substantially prevent hybridization, and
(c) a label chemically linked to (b), the labeled hybridizable nucleic acid
produced by a process of
(i) photochemically linking the nucleic acid component to the intercalator
compound and chemically linking a label to a photochemically linked
intercalator compound, or
(ii) chemically linking a label to the intercalator compound to form a
composite and photochemically linking said composite to the nucleic acid
component, wherein the nucleic acid is modified at not more than one site
per 50 nucleotide bases.
29. A labeled hybridizable nucleic acid comprising
(a) a nucleic acid component,
(b) an intercalator compound photochemically linked to the nucleic acid
component thereby modifying the nucleic acid such that the frequency of
modification along a hybridizable single stranded portion of the nucleic
acid not be so great as to substantially prevent hybridization, and
(c) a label chemically linked to (b), the labeled hybridizable nucleic acid
produced by a process of
(i) photochemically linking the nucleic acid component to the intercalator
compound and chemically linking a label to a photochemically linked
intercalator compound, or
(ii) chemically linking a label to the intercalator compound to form a
composite and photochemically linking said composite to the nucleic acid
component, wherein the nucleic acid is modified at not more than one site
per 100 nucleotide bases.
30. A kit for determining a particular polynucleotide sequence in a test
sample, comprising in one or more containers
(a) a polynucleotide probe having a base sequence complementary to the
sequence to be determined and
(b) a mono-adduct forming nucleic acid-binding ligand being chemically
linked to a detectable label moiety, said ligand being photochemically
linked to the probe or being capable of being photochemically linked to
the sample, wherein the label is selected from the group consisting of
fluorescein and phycobiliprotein.
31. A kit according to claim 30, wherein the nucleic acid-binding ligand is
an intercalator compound selected from the group consisting of acridine
dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and
quinolines.
32. A kit according to claim 31, wherein the intercalator compound is a
furocoumarin or a phenanthridine.
33. A kit according to claim 30, wherein the label is fluorescein.
34. A kit according to claim 30, wherein the label is phycobiliprotein.
35. A method according to claim 14, wherein the label is phycobiliprotein.
36. A labeled hybridizable nucleic acid according to claim 27, wherein the
linker group is selected from the group consisting of dithiobis
succinimidyl propionate and 1,4-butanediol diglycidyl ether. |
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Claims |
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Description |
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The present invention relates to a photochemical method of labelling
nucleic acids for detection purposes in hybridization assays for the
determination of specific polynucleotide sequences.
The most efficient 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 consuming and requires
experienced technical people. Recently, a non-radioactive method of
labelling DNA has been described by Ward et al, European Pat. Appl.
63,879; they use the method of nick translation to introduce biotinylated
U residue to DNA replacing T. The biotin residue is then assayed with
antibiotin antibody or an avidin containing system. The detection in this
case is quicker than autoradiography but the method of nick translation is
a highly skilled art. Moreover, biotinylation using biotinylated UTP works
only for thymine-containing polynucleotides. Use of other nucleotide
triphosphates is very difficult because the chemical derivatization of A
or G or C (containing --NH.sub.2) with biotin requires elaborate and
highly skilled organic chemists.
It is accordingly an object of the present invention to provide a
simplified system for detection of nucleic acids by hybridization assays,
which system can be easily produced and used without the disadvantages
noted hereinabove.
These and other objects and advantages are realized in accordance with the
present invention pursuant to which the nucleic acid is labeled by means
of photochemistry, employing a photoreactive nucleic acid-binding ligand,
e.g., an intercalator compound such as a furocoumarin or a phenanthridine
compound or a non-intercalator 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).
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:
##STR1##
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. Alternatively, where the labeled DNA comprises the hybridizable
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.
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 form
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, 4'-aminomethyltrioxsalen
(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
Chemistry 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
B. Phenanthridines Bloomfield et al, supra
ethidium Miller et al, supra
coralyne Wilson et al, J. Med.
Chem. 19:1261(1976)
ellipticine, ellipticine
Festy et al, FEBS
cation and derivatives
Letters 17:321(1971);
Kohn et al, Cancer Res.
35:71(1976); LePecq et
al, PNAS (USA)71:
5078(1974); Pelaprat et
al, J. Med. Chem.
23:1330(1980)
C. Phenazines Bloomfield et al, supra
5-methylphenazine cation
D. Phenothiazines "
chlopromazine
E. Quinolines "
chloroquine
quinine
F. Aflatoxin "
G. Polycyclic hydrocarbons
"
and their oxirane
derivatives
3,4-benzpyrene Yang et al, Biochem.
benzopyrene diol Biophys. Res. Comm.
epoxide, 1-pyrenyl-
82:929(1978)
oxirane
benzanthracene-5,6-oxide
Amea et al, Science
176:47(1972)
H. Actinomycins Bloomfield et al, supra
actinomycin D
I. Anthracyclinones "
rhodomycin A
daunamycin
J. Thiaxanthenones "
miracil D
K. Anthramycin "
L. Mitomycin Ogawa et al, Nucl.
Acids Res., Spec.
Publ. 3:79(1977);
Akhtar et al, Can. J.
Chem. 53:2891(2975)
M. Platinum Complexes Lippard, Accts. Chem.
Res. 11:211(1978)
N. Polyintercalators Waring et al, Nature
echinomycin 252:653(1974);
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 LePecq 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
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);
al, JACS 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)
P. Fluorenes d fluorenone
Bloomfield et al, supra
fluorenodiamines Witkowski et al,
Wiss. Beitr.-Martin-
Luther-Univ. Halle
Wittenberg, 11(1981)
Q. Furocoumarins
angelicin Venema et al, MGG,
Mol. Gen. Genet.
179;1 (1980)
4,5'-dimethylangelicin
Vedaldi et al, Chem.-
Biol. Interact. 36:
275(1981)
psoralen Marciani et al, Z.
Naturforsch 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)
______________________________________
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
[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. 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 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 .sup.3 H, .sup.35 S, .sup.32 P, .sup.125 I, and .sup.14 C. 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 (e.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,
.beta.-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 method 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 formation without interfering with
the photochemical reactivity of the furocoumarin or the biological
activity of the biotin, e.g.,
##STR2##
Other aminomethylangelicin, psoralen and phenanthridium derivatives can be
similarly reacted, as can phenanthridium halides and derivatives thereof
such as aminopropyl methidium chloride, i.e.
##STR3##
[see Hertzberg et al, J. Amer. Chem. Soc. 104: 313 (1982)]
Alternatively a bifunctional reagent such as dithiobis 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 glutaraldehyde 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 difficulties.
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 coupled to the label. Alternatively, the
psoralen can first be coupled to a label such as an enzyme and then to the
nucleic 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-hydroxy psoralen derivative with an alkyl halide,
according to the method described by J. L. DeCout and J. Lhomme,
Photochemistry Photobiology, 37, 155-161 (1983). The haloalkylated
derivative is then reacted 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 .beta.-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 comprised of two or more
individual segments interrupted by nonhomologous sequences. These
nonhomologous sequences can be linear or they can be self-complementary
and form hairpin loops. In addition, the homologous region of 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 RNA. Useful probes include linear or
circular probes wherein the homologous probe sequence is in essentially
only single stranded form [see particularly, Hu and Messing, Gene 17: 271
(1982)].
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 acids 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 which bind nucleic acids either
covalently or non-covalently. Noncovalent supports are generally
understood to involve hydrophobic bonding include naturally occurring and
synthetic polymeric materials, such as nitrocellulose, derivatized nylon,
and fluorinated polyhydrocarbons, in a variety of forms such as filters 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 immobilization of
sample nucleic acids onto the support in single stranded form. This
initial step essentially 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.
EXAMPLE 1
50 mg of N-hydroxysuccinimido biotin is dissolved in 2 ml dimethylsulfoxide
(soln 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 HCl) solution pH.about.8.
Solution (A) and (B) 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 35.degree. C. for 1 hour. The extent of
the reaction is monitored by thin layer chromatography--on silica gel
plates with a fluorescence indicators n 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 reaction. 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
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