Note: Descriptions are shown in the official language in which they were submitted.
~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 - ~
1 0 0
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
11
-
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
/ CH3
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.
12
. _ _ . . . . ... , . ... . _ . . _ _ _ _ _ _
~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
14
~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
16
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
18
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.
19
~%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.
22
