CA1155041A

CA1155041A - Fluorescent nucleic acid stains

Info

Publication number: CA1155041A
Application number: CA000371297A
Authority: CA
Inventors: Michael E. Jolley; Chao-Huei J. Wang
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1980-04-21
Filing date: 1981-02-19
Publication date: 1983-10-11
Also published as: GB2074340A; GB2074340B; DE3115278A1; FR2480943A1; DE3115278C2; BE888509A; FR2480943B1; JPS6361622B2; JPS56158944A

Abstract

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.

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.

.
: .

~.
:::

_ 7 _ 11SSQ41 __ _ _ ~ o .
~, o O ~h ~ c~ ~ ~ ~- ~D 00 ~ ~r ~ Lr~ ~ u~ r~ a) ~ ~ ~ ~
~ ~ ~ ~ o ~ ~r ~ ~ oo co ~ ~ I~ ~ ~ O O ~ O ~ O
a o O O O O O O O ~ O O O O O O O o o o . a~
a) ~
~o al ~
~ a o o K u~ o~ ~ r` ~ ~ ~ o o o ~ + ~ 10~

H

H ~ Z
~ a) a ,, ~ ~ x ~ D ~ O ~
~ ~:) ~ ~ ~ Ir) ~ ~ Ul N ~ ~1 ~ N ~I ~1 ~i U~ C~ ~
m ~ ~ ~ ~ ~ , i~i s a . .
~ .
o .~, ~
Ul F~ L~ CO O ) ~D O O ~ 1-- 0 Ll (~ ~ o ~ L~
~ ~ CJ~ ~D ~ O a~ ~ o ~ o ~ o ~ a~ co o - ~ c~

r~

~ ~ O O O OLl~ O O O O O L~ r o o o ~
~ '.-~ ~ ~ r~ o ~ ~ ~ ~ o a _ ~. .

- 8 - ~.~SS(~4~

~ o a) ~
~ ~o c~ ~ ~ Ln ~ ~ ~ ~ ~ ~ ~ ~ ~ r~ ~ o o o ~ o o o o ~ o o o o o ~I ~ o ~1 Z
oooooooooooooooooo .
H VV V V V V
~) :~
~; O

aD1) Z
a) c.~ ~ ~ ~~ ~ ~D r~ r~ ~ ~ ~ ~ ~ ~ ~I ~
_ C~ + ~
~ ~ a .
~: .~ ~

a u~ u~ Ln r a~ N ~ ~1 00 U~ N ~1 ~r d! U) N
U ~

~ _~ In O CO O O O Ln O U~ O ~ ~r o ~r co ~ In Ln ,~ ~ ~ o ~ ~ ~ ~ r~ ~ ~ ~ ~ ~ r~ r~ ~ ~ ~ ~D
~ _ ~D r~ 9 r~ D Lr~ u~

~o ~ ~ O O ~ O ~ L~ ~ Lr) O Ln In O O O U~ U~ O U~
u ~ ~ ~ ~ ~ co ~ ~ ~ ~ ~ ~ ~ ~ ~ r~ ~ N
. .

~ ~ ~ ~ ~r n ~ r- CO ~ o ~ ~ ~ ~ D r~ CO
r~
------- -ll5SOA~
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.

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.

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 --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~

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.

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.
:

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.

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.

Claims

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:

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.

2. A method of detecting nucleuc acids in a biological sample which comprises staining said acids with a fluorescent dye of the formula:

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.

3. A method of detecting nucleic acids in a biological sample which comprises staining said acids with a fluorescent dye of the formula:

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.

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.