COLORANTS FLUORESCENTS DES ACIDES NUCLEIQUES

FLUORESCENT NUCLEIC ACID STAINS

Abrégé anglais

Canada - #3763
A B S T R A C T
This invention relates to known dyes which are
fluorescent when bound to nucleic acids. This heretofore
unknown property has been shown to be useful in detecting
a variety of viruses, bacteria, yeasts, fungi, plasmids,
reticulocytes and all manner of living cells in biological
samples.

Revendications

- 12-
The embodiments of the invention in which an exclusive property
or privilege is claimed, are defined as follows:
1. A method of detecting nucleic acids in a
biological sample which comprises staining said acids with a
fluorescent dye of the formula:
<IMG>
wherein n represents an integer having a value of 0 or 1;
m represents an integer having a value of 1 or 2;
R represents a member selected from the group con-
sisting of an alkyl radical having 1 to 4 carbon atoms which
may be substituted with dialkylamino having 1 - 4 carbon atoms
in the alkyl moieties;
R1 and R2 each represent members selected from the
group consisting of hydrogen, lower alkyl and halo substituted
lower alkyl;
R3 represents a member selected from the group con-
sisting of hydrogen, lower alkyl, lower alkoxy and amino;
Z represents the nonmetallic atoms necessary to
complete a heterocyclic nucleus selected from the group con-
sisting of a benzothiazole nucleus, indolenine nucleus,
naphthothiazole nucleua, benzoselenazole nucleus, benzoxazole
nucleus, quinoline nucleus, and pyridine nucleus any of which
may be substituted with lower alkyl, halo, nitro, and dialkyl
amino; and
X represents an anion.

- 13 -
2. A method of detecting nucleuc acids in a
biological sample which comprises staining said acids with
a fluorescent dye of the formula:
<IMG>
wherein n represents an integer having a value of 0 or 1;
R represents a member selected from the group con-
sisting of an alkyl radical having 1 - 4 carbon atoms which
may be substituted with dialkylamino having 1 - 4 carbon atoms
in the alkyl moieties;
R1 and R2 each represent members selected from the
group consisting of hydrogen, lower alkyl and halo substituted
lower alkyl;
R3 represents a member selected from the group con-
sisting of hydrogen, lower alkyl, lower alkoxy and amino;
Z represents the nonmetallic atoms necessary to
complete a heterocyclic nucleus selected from the group con-
sisting of a benzothiazole nucleus, indolenine nucleus,
naphthothiazole nucleus, benzoselenazole nucleus, benzoxazole
nucleus, quinoline nucleus, and pyridine nucleus any of which
may be substituted with lower alkyl, halo, nitro, and dialkyl
amino; and
X represents an anion.

- 14 -
3. A method of detecting nucleic acids in a
biological sample which comprises staining said acids with
a fluorescent dye of the formula:
<IMG>
wherein R represents a member selected from the group con-
sisting of an alkyl radical having 1 - 4 carbon atoms which
may be substituted with dialkylamino having 1 - 4 carbon atoms
in the alkyl moieties;
R1 and R2 each represent members selected from the
group consisting of hydrogen, lower alkyl and halo substituted
lower alkyl;
Z represents the nonmetallic atoms necessary to
complete a heterocyclic nucleus selected from the group con-
sisting of a benzothiazole nucleus, indolenine nucleus,
naphthothiazole nucleus, benzoselenazole nucleus, benzoxazole
nucleus, quinoline nucleus, and pyridine nucleus any of which
may be substituted with lower alkyl, halo, nitro and dialkyl
amino; and
X represents an anion,
4. A method according to Claim 1 wherein the
fluorescent dye is 1-?-dimethylaminopropyl-4-p-dimethylamino-
styrylquinolinium iodide.
5. A method according to Claim 1 wherein the
fluorescent dye is 3-?-dimethylaminopropyl-2-p-dimethylamino-
styrylbenzothiazolium iodide.
6. A method according to Claim 1 wherein the
fluorescent dye is 3-?-dimethylaminopropyl-5-chloro-2-di-
methylaminostyrylbenzothiazolium iodide.

- 15 -
7. A method according to Claim 1 wherein the
fluorescent dye is 3-?-dimethylaminopropyl-2-p-dimethylamino-
styryl-.alpha.-naphthothiazolium iodide.
8. A method according to Claim 1 wherein the
fluorescent dye is 3-?-dimethylaminopropyl-2-p-dimethylamino-
styrylbenzoselenazolium iodide.
9. A method according to Claim 1 wherein the
fluorescent dye is 3-?-dimethylaminopropyl-5-methyl-2-p-
dimethylaminostyrylbenzothiazolium iodide.
10. A method according to Claim 1 wherein the
fluorescent dye is 1-?-dimethylaminopropyl-4-p-dimethylamino-
styrylpyridinium iodide.
11. A method according to Claim 1 wherein the
fluorescent dye is 1-methyl-2-dimethylamino-4-p-dimethyl-
aminostyrylpryidinium iodide.

Description

5S~4~
sackground Of The Invention
1. Field Of The Invention
This invention relates generally to the technique
of fluorescence microscopy which has developed recently into
5 a valuable tool for biological research. Fluorescence micro-
scopy is an extraordinarily sensitive method for detecting
minute quantities of substances which can be stained with dyes
that fluoresce when excited with incident light. More
particularly, this disclosure relates to a method of detecting
10 a variety of organisms in biological samples. The detection
of said organisms is facilitated by the nucleic acid staining
properties of the disclosed fluorescent dyes.
Description Of The Prior Art
It is apparent from the scientific literature that
15 a variety of compounds have been used to selectively stain
nucleic acids. Von Bertalanffy and Bickis, J. Histochem.,
Cytochem., Vol. 4, pp. 481-493, (1956), reported that acridine
orange could be used for the identification of cytoplasmic
basophilia (RNA) by fluorescence microscopy. In particular,
20 they found that acridine orange enables identification of
basophilic cytoplasmic inclusions in the supravital state,
and that the red fluorescent cytoplasmic inclusions corres-
pond to those stained with the toluidine blue technique and
shown by ribonuclease to consist mainly of RNA.
Rossell, et al reported in Nature, Vol. 253, p. 461,
(1975) that 4'-6-diamidino-2-phenylindole (DAPI) has been shown
to possess useful DNA binding properties, Specifically, they
found that DAPI can be used as a hi~hly specific fluorescent
stain for both nuclear and mitochondrial DNA in yeast.
Hilwig and Gropp in Experimental Cell Research 75,
pp 122-126 (1972) discuss a simple and direct fluorescense
staining procedure using a benzimidazole derivative (identified
as 33258 Hoechst) to visualize chromosomal segments of heter-
chromatin and (in the mouse) sites of repetitious DNA.
While the prior art dyes appear to be widely used
in fluorescent microscopy, histology and fluorescence micro-

- 2 - ~5504~
fluorometry, they have undesirable properties, in terms of
exci-tation/emission spectra, quantum yield~ and background
fluorescence, which have prompted investigators to search
for alternative fluorescent nucleic acid dyes. It is apparent~
therefore, that there is a need for nucleic acid specific
dyes, excitable at visible wavelengths, which are essentially
nonf1uorescent in their free state~ but are highly fluores-
cent when bound to nucleic acids. It has been found that the
dyes herein disclosed possess these properties.
The compounds useful in the present disclosure have
been known generally as dyes from U. S. Patent 3,833~863 and
Defensive Publication T88 9016. However, neither reference
disclosed nor suggested the novel use.
Summary Of The Invention
Specifically, this disclosure is directed to a novel
method of detecting nucleic acids which comprises staining
said acids with a fluorescent dye of the formula;
R-1~3( =CH-C~C- ( C~l=C~ ) m{~ ~ ~~N ~
wherein n represents an integer having a value of 0 or l;
m represents an integer having a value of l
or 2;
R represents a member selected from the group
consisting of an alkyl radical having l to 4 carbon atoms
which may be substituted with dialkylamino having l - 4
carbon atoms in the alkyl moieties;
Rl and R2 each represent members selected from the
group consisting of hydrogen, lower alkyl and halo substituted
lower alkyl;

- 3 - ~SSQ~
R3 represents a member selected from the group
consisting of hydrogen, lower alkoxy, lower alkyl and amino;
Z represents the nonmetallic atoms necessary to
complete a heterocyclic nucleus selected from the group con-
sisting of a benzothiazole nucleus, indolenino nucleus,naphthothiazole nucleus, benzoselenazole nucleus, benzoxazole
nucleus, quinoline nucleus, and pyridine nucleus any of which
may be substituted with lower alkyl, halo, nitro and dialkyl
amino; and
X represents an anion.
Description Of The Preferred Embodiment
The disclosed dyes are essentially nonfluorescent
in aqueous solutions, but they show marked enchancements when
bound to double-stranded DNA. Some of these dyes do not
exhibit this enhancement when bound to siligle-stranded RNA;
however, a significant increase in fluorescence is apparent
when most of the dyes are bound to RNA. Because of the ab-
sence of fluorescence in the free-state, large auantities of
the dye may be employed without having to separate or remove
excess reagent. This property is important in some appli-
cations where poor absorption of the dye occurs. In
addition, because of the high fluorescence quantum yield of
some of the dyes, small quantities of dye may be us,ed if
desired.
The`following dyes were prepared and tested for the
clalmed utility.
:

- 4 - ~ ~55~
Dye No. Chemical Name
1 3-y-dimethylaminopropyl-2-p-dimethylamino-
styrylbenzothiazolium iodide
2 3-y-dimethylaminopropyl-2-_-dimethylamino-
styrylbenzoxazolium iodide
3 1-y-dimethylaminopropyl-2-~-dimethylamino-
styryl-3,3-dimethyl-3H-indolium iodide
- 4 3-methyl-6-dimethylamino-2-_-dimethylamino-
styrylbenzothiazolium iodide
3-~-dimethylaminoethyl-2-p-dimethylamino-
styrylbenzothiazolium iodide
6 3-y-dimethylaminopropyl-2-p-diethylamino-
styrylbenzothiazolium iodide
7 3-y-dimethylaminopropyl-5-chloro-2-_-dimethyl-
aminostyrylbenzothiazolium iodide
8 3-y-dimethylaminopropyl-2-p-dimethylamin
styryl-~-naphthothiazolium iodide
9 3-y-dimethylaminopropyl-2-_-dimethylamino-
styrylbenzoselenazolium iodide
3-y-dimethylaminopropyl-5-methyl-2-p-di-
methylaminostyrylbenzothiazolium lodide
11 3-~-diethylaminoethyl-2-p-dimethylamino-
styrylbenzothiazolium iodide
12 3-Y-dimethylaminopropyl-2-p-(N-2-chloro-
ethyl-N-ethyl)-aminostyrylbenzothia-
zolium iodide
: 13 3-y-dimethylaminopropyl-2-p-N,N-bis-(2-
chloroethyl)-amino--methylstyrylbenzo-
thiazolium iodide
14 3-y-dimethylaminopropyl-2-p-N,N-bis-(2-
chloroethyl)-aminostyrylbenzothiazolium
iodide
3-(~-pyrrolidinoethyl)-2-p-dimethylamino-
styrylbenzothiazolium iodide
16 3-(~-morpholinoethyl)-2-p-dimethylamino-
styrylbenzothiazolium iodide
17 3-y-dimethylaminopropyl-2-(4-p-dimethyl-
aminophenyl-1,3-butadi.enyl)-benzothia-
zoliur iodide

~15504~
Dye No. _ Chemical Name
18 3-y-dimethylaminopropyl-2-(4-_-dimethyl-
aminophenyl-1,3-butadienyl)-benzoxa-
zolium iodide
19 3-y-dimethylaminopropyl-2-(4-p-dimethyl-
aminophenyl-1,3-butadienyl)-5-methyl-
benzothiazolium iodide
3-y-dimethylaminopropyl-2-(4-p-dimethyl-
aminophenyl-1,3-butadienyl)-~-naphtho-
thiazolium iodide
21 3-~-dimethylaminopropyl-2-(4-p-dimethyl-
aminophenyl-1,3-butadienyl-5-chloro-
benzothiazolium iodide
22 3-y-dimethylaminopropyl-2-(A-p-dimethyl-
aminophenyl-1,3-butadienyl)-6-nitro-
benzothiazolium iodide
23 3-y-dimethylaminopropyl-2-(4-p-dimethyl-
aminophenyl-1,3-butadienyl)-benzo-
selenazolium iodide
24 3-y-diethylaminopropyl-2-(4-p-dimethyl-
aminophenyl-1,3-butadienyl)-benzo-
thiazolium iodide
1-y-dimethylaminopropyl-4-_-dimethyl-
aminostyrylquinolinium iodide
26 1-y-dimethylaminopropyl-2-p-dimethyl-
aminostyrylquinolinium iodide
27 1-y-dimethylaminopropyl-4-(4-p-dimethyl-
aminophenyl-1,3-butadienyl)-quinolinium
iodide
28 1-~-dimethylaminopropyl-4-p-dimethylamino-
styrylpyridinium iodide
29 3-propyl-2-p-dimethylaminostyrylbenzo-
thiaæolium iodide
1-ethyl-4-_-diethylaminostyrylquinolinium
iodide
31 Anhydro-3-y-sul~opropyl-2-_-dimethylamino-
styrylbenzothiazolium hydroxide
32 3-~-carboxyethyl-2-_-dimethylaminostyryl-
benzothiazolium iodi..lr~

- 6 - 1~5S~4~
Dye No. _ _ Chemical Name
33 1-methyl-2-dimethylamino-4-_-dimethylamino-
styrylpyridinium iodide~
34 1-methyl-2-dimethylamino-4-_-diethylamino-
styrylpyridinium iodide
1-ethyl-2-diethylamino-4-p-diethylamino-
styrylpyridinium iodide
36 1-methyl-2-dimethylamino-4-~-N,N-bis-
(2-chloroethyl)-aminostyrylpyridinium
iodide
37 1-methyl-2-dimethylamino-4-~-N,N-bis-
(2-chloroethyl)-amino-o-methyl-
styrylpyridinium iodide
- 38 1-ethyl-2-diethylamino-4-_-dimethylamino-
styrylpyridinium iodide
.
The above-identified dyes were irradiated to
determine excitation wavelengths and the reslllting fluorescent
emission. Then, samples of DNA and RNA were stained with
each dye. The samples were irradiated at the appropriate
wavelen~th and the fluorescent enhancement was observed.
The results are tabulated in Table I.
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g
All of the above data were obtained using a
Perkin-Elmer MPF 43-A Fluorescence Spectrophotometer, using
0.1 ~M of dye in 3 ml of 10 n~ phosphate buffered saline
(PBS), pH 7.3.
The ratios depicting fluorescence enhancement were
obtained by taking the ratio of the ~luorescence intensity
or a dye solution containing 100 micrograms per ml of calf
thymus type I DNA and yeast type III RNA to that of the free
dye solution.
The relative fluorescence is simply obtained from
the ratio of the fluorescence intensity of a dye solution
containing 100 micrograms per ml of calf thymus type I DNA
to that of Dye 1. solution containing the same amount of DNA.
The data are uncorrected for instrument response at the dif-
ferent wavelengths.
EXAMPLE 1
5 ~1 of a solution of dye 1 (2 mg/ml in o.9%
saline) was added to 100 ~1 of fresh whole blood (EDTA) The
mixture was shaken and a wet slide immediately prepared for
examination under the fluorescence microscope (540 nm broad-
band excitation filter, 590 nm long pass emission filter;
400 x magnification). The nuclei of the peripheral blood
leukocytes (PBLs) were observed and stained an intense orange
against a black background. In addition, moderately intense-
ly stained platelets were observed. No staining was visiblein the erythrocytes or in the cytoplasm of the PBLs.
EXAMPLE 2
20 ~1 of a solution o~ dye 1 (2 mg/ml in 0,9~
saline) was added to 2 ml o~ a suspension of E~ coli (108 _
cells/ml). The mixture was vortexed and a wet slide
prepared and examined as described in Example 1. The bacteria
were observed as brilliant orange rods against a black
background~
:

~115S041
-- ~o --
EXAMPLE 3
To 2 ml of a suspension of E. coli (~ 10 cells
per ml) was added 20 ~1 of a 2 mgjml solution of dye 25.
The mixture was shaken and a wet slide prepared~ The slide
was observed as described in Example 1~ The bacteria were
observed as brilliant red rods on a black background~
AMPLE 4
To 100 ~1 of phage suspensions (PICM~ 5 x 10
pfu/ml; T6, 4 x 10 pfu/ml; 2 x 10 pfu/ml~ was added 10 ~1
of a solution of dye 1 (1.6 mg/ml in 0.9~ saline; sterile
filtered through a 0.3 um filter). The mixture was shaken
and a wet slide prepared. The slide was observed under the
fluorescence microscope as described in Example 1. PICM and
T6 could be observed as small pinpoints of orange light
against a black background.
EXAMPLE 5
Stock solutions of DNA and dye I in 50 nM phosphate
buffer pH 6.8 were mixed so that the final concentrations
were 1-100 nm DN~ base pairs and 1 ~M dye in a final volume
of 2 ml. The fluorescence of the solutions were measured
on a Perkin-Elmer MPF 43A, Excitation was at 555 nm ~4 nm
bandpass); emission was at 605 nm (4 nm bandpass). A linear
curbe of fluorescence versus DNA concentration was obtained
over this range.
:
EXAMPLE 6
To 0.5 ml of a solution of dye 28 (50 ~g~ml in
0.9% saline; sterile filtered through a 0.3 ~m filter) was
added 5 ~1 of fresh whole blood ~EDTA). The mixture was
shaken and a wet slide prepared for examination under
fluorescence microscope (450 - 490 nm broadband excitation,
530 nm long pass emission filter; 400 x magnification).
The nuclei of the peripheral blood leukocytes were observed
stained bright orange, the endoplastic reticulum stained
yellow, the cytoplasm and the membrane stained green~ The
platelets were stained orange. Erythrocytes and reticulocytes

155QA~
can easily be distinguished by the intensely stained
yellow-orange reticulum of the reticulocytes and the weakly
stained greenish membrane of erythrocytes.
EXAMPLE 7
The procedures described in Example 6 were re-
peated using a solution of dye 33. The nuclei and endo-
plastic reticulum of the peripheral blood leukocytes were
stained intensely yellow, the cytoplasm and the membrane
were stained intensely green-yellow. Erythrocytes and
reticulocytes can also be distinguished easily by the
intensely stained yellowish reticulum of the reticulocytes
and the plain green-yellow stained membrane of erythrocytes.
It should be evident from the foregoing examples,
that dyes exhibiting the claimed utility can be used to
detect an almost limitless variety of microscope organisms
in biological samples. In addition to obvious uses in
microfluorescence cytology, these dyes could be used in flow
cytofluorimetry instruments to screen urine and water samples
for elevated levels of bacteria. Whole blood could also
be analyzed to provide a profile of components because these
dyes will fluorescently stain plasmids, reticulocytes, leuco-
cytes and platelets.