SUBSTITUTED UNSYMMETRICAL CYANINE DYES WITH SELECTED PERMEABILITY

FIELD OF THE INVENTION

The invention relates to fluorescent dyes for nucleic acids In particular, the invention relates to dyes derived from unsymmetπcal cyamne dyes havmg defined substituents on the quinolinium or pyπdinium ring system, where the substituents serve to mcrease or decrease the permeability, selectivity and bmdmg affinity of the nucleic acid sta s The subject dyes, which form a fluorescent complex m combination with nucleic acids, can be used m analyzing a wide range of materials, including biological and environmental samples

BACKGROUND INFORMATION

In many fields of life sciences research, including biological, biomedical, genetic, fermentation, aquaculture, agricultural, forensic and environmental research, there is a need to identify nucleic acids, qualitatively and quantitatively, in pure solutions and in biological samples Such applications require a fast, sensitive, and selective methodology that can detect minute amounts of nucleic acids in a vanety of media, whether or not the nucleic acid is contained in cells

Although certain unsymmetπcal cyamne dyes were first descnbed before the genetic role of nucleic acids was established (Brooker, et al , J AM CHEM SOC 64, 199 (1942)), some unsymmetπcal cyamne dyes are now known as effective fluorescent stams of DNA and RNA The compound sold as Thiazole Orange has particular advantages in reticulocyte analysis (U S Patent No 4,883,867 to Lee, et al (1989)) or m preferentially staining bloodborne parasites (U S Patent No 4,937, 198 to Lee, et al (1990)) Thiazole Orange readily stams many mammalian cells, yet does not effectively stain some eukaryotic cells

Attachment of various cyclic structures to the pyrdinium or quinolinium ring system of the unsymmetπcal cyamne dye was found to make the nucleic acid stains highly permeant to gels and a wider vanety of cell types, including both Gram-positive and Gram-negative bacteπa, yeasts, and eukaryotic cells as well as prokaryotic cells (U S Patent No 5,436, 134 to Haugland, et al (1995), FLUORESCENT ASSAY FOR BACTERIAL GRAM REACTION (Ser No 08/146,328 to Roth et al filed 1 1/01/93), FLUORESCENT VIABILITY ASSAY USING CYCLIC-SUBSTITUTED

UNSYMMETRICAL CYANINE DYES (Ser No 08/148,847 to Millard, et al filed 1 1 08/93), and U S Patent No 5,445,946 to Roth et al (1995)), and International Publication No WO 94/24213 (Corresponding to PCT application 94/04127)

Attachment of a cationic side cham at the nitrogen of the pyndimum or quinolinium πng system of the unsymmetπcal cyamne dyes, on the other hand, was shown to make the stams relatively impermeant to all cells, except cells, particularly mammalian cells, where cell membrane integπry was destroyed, as descnbed m UNSYMMETRICAL CYANINE DYES WITH CATIONIC SIDE CHAINS (U S Patent no 5,321,130 to Yue et al (1994)) A second type of dye, in which a dye monomer is attached at the nitrogen of the quinolinium or pyndinium ring system to form dimenc

compounds as descnbed m DIMERS OF UNSYMMETRICAL CYANINE DYES (PCT 92/07867) and DIMERS OF UNSYMMETRICAL CYANINE DYES CONTAINING PYRIDINIUM MOIETIES (U S Patent No 5,410,030 to Yue, et al (1995)) that are also relatively impermeant to all cells unless the cell membrane has been disrupted Although these impermeant dyes were found to have the further advantage of mcreased bmdmg affinity for nucleic acids, resultmg m mcreased sensiUvity for detection of cell free nucleic acids, a number of these dyes were also found to have a number of disadvantages for some applications, mcludmg a slow rate of equilibπum bmdmg, electrostaUc attraction to glass surfaces, moderate salt sensitivity, reduced photostabihty, lower quantum yield, relatively lower sensitivity of detection of nucleic acids in gels and in solutions, and limited permeability to dead prokaryotic cells

The dyes of the present vention are unsymmetncal cya ne dyes containing a defined substituent on the pyndimum or quinolinium ring system or a substituent immediately adjacent to the nitrogen atom of the pyndimum or quinolinium πng that modifies the permeability, selectivity and affinity of the dye for nucleic acids Members of this class of dyes are more effective in detection of cell membrane integnty and m the staining or detection of nucleic acids, mcludmg DNA and RNA, in gels and in solutions, and in living and dead cells Dyes substituted at the posiUon adjacent to the ring nitrogen generally have unexpectedly higher quantum yields than dyes not substituted at that position In addiUon, the nng substituent is easily modified, particularly by inclusion of an appropπate heteroatom m the substituent, to allow selectable alteration of the permeability and affinity of the dyes Furthermore, by simple synthetic modification, a family of dyes having absorption and emission spectral properties that cover most of the visible and near-infrared spectrum can be prepared Selection of an appropπately substituted dye enhances the sensitivity of analysis of nucleic acids utilizing a vanety of techniques

DESCRIPTION OF DRAWINGS

Figure 1 The fluorescence excitation and emission spectra for Dye 309 in the presence of ds calf thymus DNA Note the lower intensity absorbances in the UV region of the excitation spectrum, indicating that fluorescence can be generated by excitation at those wavelengths, albeit at lower fluorescence yields

Figure 2 A multiple labeling experiment using the dyes of the present mvention Nucleic acid polymers are generated that are labeled with a detection reagent that is a fluorophore, avidin, streptavidin or other hapten (X, Y, and Z) After separation, the resultmg bands are visualized and quantitated by staining with a dye of the present mvention The bands can be individually identified by treatment with the appropnate reagent, such as biotin, or an antibody (X*, Y*, or Z*) This technique is generally descnbed in Example 33

Figures 3A and 3B Lmear fluorescence response as a function of DNA concentration, as descnbed m Example 21

The assay is lmear from 25 pg/mL (see mset) to 1000 ng/mL

Figure 4 Lmear fluorescence response as a function of oligonucleotide concentration, as descnbed in Example 22 The assay is lmear from an oligonucleotide concentration of 100 pg/mL (see mset) to 1 μg/mL

Figures 5 A and 5B Lmear fluorescence response as a function of cell number, as descnbed m Example 38 Standard concentration plots are shown for both NH/3T3 cells and PX3 cells

Figure 6 Fluorescence emission as a function of nucleic acid type, as descnbed m Example 40

Figure 7 Fluorescence emission as a function of dye-loading, as descnbed in Example 40

Figures 8 A, 8B Analysis of bacteπal metabolic activity, as descnbed in Example 45 The cluster of data m Figure 8 A represents metabo cally active bactena The signal cluster in Figure 8B represents metabo cally quiescent bactena

Figure 9 Analysis of a cell suspension by flow cytometry, as descnbed in Example 47 A lmear relationship exists between the distπbution of cells m the two regions and the actual percentage of live cells m the sample, as shown m the inset figure

Figures 10A, 10B Analysis of cell cycle distnbution using flow cytometry, as descnbed in Example 48 Figure 10A shows clusters of signals correspondmg to cells m the Gl , S and G2 phases of the cell cycle Figure 10B depicts a histogram showmg the distnbution of cells among the Gl , S and G2 compartments of the cell cycle

Figure 1 1 A, 1 IB Analysis of cell proliferation usmg bromodeoxyundine labeling followed by bivaπate staining, as descnbed Example 49 Figure 1 1 A shows clusters of signals correspondmg to cells m the GO/G 1 , S and G2 phases of the cell cycle Figure 11 B depicts a histogram showmg the distnbution of cells among the GO/G 1 , S and G2 compartments of the cell cycle

SUMMARY OF THE INVENTION AND DESCRIPTION OF PREFERRED EMBODIMENTS

The substituted unsymmetncal cyamne dyes of the mvention are virtually non-fluorescent when diluted m aqueous solution When bound to nucleic acid polymers such as DNA and RNA, however, the resultant dye-nucleic acid complex becomes extremely fluorescent upon illummation The dyes of the present mvention label nucleic acids in a wide vanety of samples, particularly in aqueous solutions, electrophoretic gels, and a wide vanety of cells, mcludmg microorganisms

Dye Structure

The dyes of the invention compnse 1 ) a first heterocyclic nng system that is a substituted benzazolium ring,

2 'mdging methine and 3) a second heterocyclic nng that is a pyndimum or quinolinium πng system, one or more po _^ 'ions of which may be substituted by a TAIL that contains at least one heteroatom The first and second nng systems are optionally further substituted by a vanety of substituents, as descnbed below

TAIL

TAIL is a heteroatom-containmg side cham, that is descnbed by the formula LINK-SPACER-CAP LINK is the linking moiety by which TAIL is attached to the core structure of the dyes of the present mvention SPACER is a covalent linkage that connects LINK to CAP CAP is the portion of TAIL that possesses a heteroatom component

LINK is a smgle covalent bond, an ether linkage (-0-), a thioether linkage (-S-), or an amine Imkage (-NR ²⁰ -) In each embodiment, LINK forms the attachment between the dye core structure and SPACER When LINK is an amine, the amine substituent (R ²⁰ ) is optionally H, such that LINK = -NH- Alternatively, R ²⁰ is a lmear or branched alkyl having 1 -8 carbons In another embodiment of the mvention, R ²⁰ is -SPACER'-CAP', yielding a TAIL having the formula

where SPACER' and CAP', respecUvely, may be the same as or different from SPACER and CAP, and are selected from the same alternatives defined for SPACER and CAP, respectively For the sake of simplifying the descnpUon,

SPACER and CAP are defined with the understanding that a descnpUon of SPACER mcludes SPACER', and a descnpUon of CAP mcludes CAP'

SPACER is a covalent linkage that joms LINK and CAP SPACER is a lmear, branched, cyclic, heterocyclic, saturated or unsaturated arrangement of 1-16 C, N, P, O or S atoms Alternatively, SPACER is a smgle covalent bond, such that both LINK and SPACER are not simultaneously smgle covalent bonds Preferably, the SPACER linkage must beg and end with a carbon atom Typically, if SPACER consists of a single atom, it is required to be a carbon atom, so that the first and last atom in SPACER (m this specific instance, they are the same atom) is a carbon The 1-16 atoms making up SPACER are combmed using any appropπate combmation of ether, thioether, amine, ester, or amide bonds, or smgle, double, tπple or aromaUc carbon-carbon bonds, or phosphorus-oxygen bonds, or phosphorus-sulfur bonds, or nitrogen-nitrogen bonds, or nitrogen-oxygen bonds, or aromaUc or heteroaromaUc bonds SPACER is further substituted by hydrogen to accommodate the valence state of each atom in SPACER

Generally, the atoms of SPACER are arranged such that all heteroatoms in the lmear backbone of SPACER are separated by at least one carbon atom, and preferably separated by at least two carbon atoms Typically, SPACER is 1 -

6 carbon atoms in a linear or branched saturated cham In one embodiment of the mvention, SPACER incorporates a 6- membered aromatic nng (phenylene linkage) In another embodiment of the mvention, SPACER incorporates a 5- or 6- membered heteroaromaUc nng, wherein the heteroatoms are O, N, or S Alternatively, SPACER incorporates amide linkages, ester linkages, simple ethers and thioethers, and amines m a lmear arrangement, such as -CH ₂ -CH ₂ -(C=0)- NH-CH ₂ -CH ₂ -CH ₂ - Preferably, SPACER is a lmear cham composed of sequential methylene groups (-(CH ₂ ) _k -, where k = l-8)

LINK and SPACER, in combmation, serve to attach a heteroatom-containing group, CAP, to the dye core structure CAP may contain oxygen, sulfur or nitrogen, according to the formulas -O-R ²¹ , -S-R ²¹ , -NR ²¹ R ²² , or -N ⁺ R ²¹ R ²² R ²³ Ψ ^" The substituents R ²¹ , R ²² , and R ²³ are mdependently H, or a lmear or branched alkyl or cycloalkyl having 1 -8 carbons Where any of R ²¹ , R ²² and R ²³ are alkyl or cycloalkyl, they are optionally further substituted by halogen, hydroxy, alkoxy having 1 -8 carbons, amino, carboxy, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1 -8 carbons, ammo, aminoalkyl havmg 1 -8 carbons, or carboxyalkyl having 1 -8 carbons In another embodiment of the mvention, one or more of R ²¹ , R ²² and R ²³ , taken m combmation with SPACER forms a 5- or 6-membered nng that is aromatic, heteroaromaUc, a cyclic or heteroahcyclic πng When the 5- or 6- membered nng is heteroaromaUc or heteroalicychc, the nng contains 1 -3 heteroatoms that are O, N or S Alternatively, one or more of R ²¹ , R ²² , and R ²³ , taken m combmation with R ²⁰ and SPACER, forms a 5- or 6-membered nng that is aromatic, heteroaromatic, ahcyclic or heteroalicychc nng, as descnbed above Preferably, R ²¹ , R ²² are hydrogen, or alkyls hav g 1 -8 carbons R ²³ is typically H or alkyl havmg 1 -8 carbons When CAP is ->TR ²¹ R ²² R ²³ ψ", the substituents R ²¹ , R ²² and R ²³ are typically not hydrogen, so that the positive charge present on the ammonium nitrogen is not subject to equi bnum neutralization in aqueous solutions

When CAP is -NT-l^R^R ²³ Ψ ^" , the biologically compatible countenon Ψ ^" balances the positive charge present on the CAP nitrogen, which is a quaternary ammonium salt As used herein, a substance that is biologically compatible is not toxic as used, and does not have a substantially deletenous effect on biomolecules Examples of Ψ' mclude, among others, chlonde, bromide, iodide, sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate, tefrafluoroborate, tetraarylbonde, mtrate and amons of aromatic or aliphatic carboxylic acids Preferred Ψ" countenons are chlonde, iodide, perchlorate and various sulfonates

Additionally, there are several embodiments of the present mvention wherein CAP incorporates a cyclic structure In these embodiments, CAP typically incorporates a 4- to 10-membered nng, preferably a 5- or 6-membered nng, that contains at least one nitrogen atom The nitrogen atom incorporated within the cyclic structure is optionally substituted by R ²³ to give an ammonium salt Where CAP incorporates a cyclic structure, the cyclic structure optionally mcludmg an additional heteroatom (typically oxygen or sulfur) Specific versions of CAP mclude, but are not limited to, those listed in Table 1

Table 1 Examples of specific CAP moieties

CAP is preferably -NR ²¹ R ²² or -N ⁺ R^R∞R ²³ Ψ ^" , where R ²¹ , R ²² , and R ²³ are alkyls havmg 1 -6 carbons More preferably CAP is -N(CH ₃ ) ₂ or -N ⁺ (CH ₃ ) ₃ Ψ ^"

Preferably TAIL contains 6-10 non-hydrogen atoms, mcludmg LINK and CAP

Selected examples of TAIL are listed in Table 2 For each TAIL, the identities of LINK, SPACER and CAP are specified Where R ²¹ , R ²² , or R ²³ combmed with either R ²⁰ or SPACER, the combmation is mdicated m the table

Table 2: Specific examples of TAIL moieties

to

Core Structure

The core structure of the dyes of the present mvention are descnbed by the formula

where the substituted benzazolium πng system on the left is linked by a methine bπdge to the πght-hand pyndimum or quinolinium nng system One or more substituents on the core structure is optionally a TAIL

Although R ¹ on the benzazolium nng system is usually H, incorporation of one or more non-hydrogen substituents R ¹ can be used to fine tune the absorption and emission spectrum of the resulting dye The benzazole may contam more than one substituent R ¹ , which may be the same or different (t = 1 -4) Each R ¹ is optionally an alkyl group havmg from 1 -6 carbons, or a tnfluoromethyl, or a halogen, or an alkoxy havmg 1 -6 carbons Typically, each compound contains no more than one R ¹ that is not H Preferably, R ¹ is H or alkoxy, more preferably each R ¹ is H

The substituent R ² is an alkyl group havmg 1 -6 carbons, preferably methyl or ethyl, more preferably methyl

The counteπon Ψ ^" is a biologically compatible ion, as descnbed above Preferred Ψ ^" countenons are chlonde, iodide, perchlorate and vanous sulfonates

X is one of O, S, Se or NR ¹⁵ , where R ¹⁵ is an alkyl group havmg 1-6 carbons Alternatively, X is CR ¹⁶ R ¹⁷ , where R ¹⁶ and R ¹⁷ , which may be the same or different, are mdependently H or alkyl groups havmg 1 -6 carbons, or the carbons of R ¹⁶ and R ¹⁷ taken combmation complete a five or six membered saturated nng When X is CR ¹⁶ R ¹⁷ , R ¹⁶ and R ¹⁷ are typically methyls Preferably, X is O or S, more preferably X is S

The two heterocyclic nng systems are linked by 1 , 3 or 5 methine (-CH=) groups such a way as to permit extensive electronic delocalization The number of methine groups between the heteroaromaUc rings influences die spectral properties of the dye Preferably n = 0 or 1 , more preferably n = 0

The N-bound substituent R ⁵ is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group havmg 1 -6 carbons Alternatively, R ⁵ is a cyclic substituent or a TAIL Typically R ⁵ is an alkyl havmg 1 -6 carbons, preferably 1 -2 carbons, or R ⁵ is a cyclic substituent Alternatively, R ⁵ is a TAIL Typically, when R ⁵ is a TAIL, the SPACER moiety incorporates a phenylene linkage

When R ⁵ is a cyclic subsUtuent, the cyclic substituent is a saturated or unsaturated, substituted or unsubstituted nng system with 2-16 nng carbon atoms m 1-2 ahcyclic, heteroahcvclic, aromatic, or heteroaromatic rmgs containmg 1- 4 heteroatoms (wherein the hetero atoms are O, N or S) that is directly bonded to the pyndimum or quinolinium nng system by a smgle bond Ahcyclic nng systems are either linked or fused Typically R ⁵ is an aryl, a heteroaryl, or a cycloalkyl havmg 3-10 carbons, more typically an aryl or heteroaryl group Typically the aryl is a phenyl or naphthyl group, and the heteroaryl substituent is a 5- or 6-membered heteroaromatic πng, wherein the heteroatom is O, N or S Examples of ahcyclic and hetroahcychc substitutents are subsututed or unsubstituted cyclohexyls, cyclohexenyls, morpholinos, pipendmyls and piperazinyls Examples of aromatic and heteroaromatic cyclic substituents mclude substituted or unsubstituted naphthyls, phenyls, thienyls, benzothiazolyls, furanyls, oxazolyls, benzoxazolyls, and pyπdmyls Substituents on such cyclic substitutents are mdependently hydrogen, halogen, alkyl, perfluoroalkyl, a mo, alkylammo, dialkylammo, alkoxy or carboxyalkyl, each alkyl group havmg 1 -6 carbons Preferred cyclic subsituents are substituted or unsubstituted naphthyl, phenyl, thienyl, morpholmo, and cycloalkyl havmg 3-10 carbons, more preferably substituted or unsubstituted phenyl

The second nng system contains a nng fragment Y that is -CR ³ =CR ⁴ -, with subscπpts p and m equal to 0 or 1 , such that p + m = 1 For all embodiments, the nng contains a 6 membered pyndinium-based heterocycle according to one of these formulations

or

In preferred embodiments of the invention, m = 1 and p = 0 ("4-pyndmιums" and "4-quιnohnιums")

The nng substituents R ³ and R ⁴ are mdependently H, or a halogen, or an alkyl, alkenyl, polyalkenyl, alkynyl or p yalkynyl group havmg 1-6 carbons R ³ and R ⁴ are also optionally and mdependently -OR ⁸ , -SR ⁸ , -(NR ⁸ R ⁹ ), where

R ⁸ and R ⁹ , which can be the same or different, are mdependently H, alkyl groups havmg 1-6 carbons, 1-2 ahcyclic or aromaUc rmgs, or R ⁸ and R ⁹ taken m combmation are -(CH ) ₄ - or -(CH ₂ ) ₅ - to give a 5- or 6-membered nng Additionally, R ³ and R ⁴ are optionally and mdependently -OS0 ₂ R ¹⁹ where R ¹⁹ is alkyl havmg 1-6 carbons, or perfluoroalkyl havmg 1 -6 carbons, or aryl

The πng substituents R ⁶ and R ⁷ are optionally any substituent defined for R ³ and R ⁴ , with the excepUon of -OS0 ₂ R ¹⁹ Alternatively, R ⁶ and R ⁷ taken m combmation are -(CH ₂ ) _V - where v = 3 or 4, forming a fused 5- or 6- membered nng, or R ⁶ and R ⁷ , taken m combmation form a fused 6-membered aromatic πng

Alternatively, any of R ³ , R ⁴ , R ⁶ or R ⁷ could be a cyclic substituent, as defined earlier for R ⁵ Preferred πng substituents are mdependently H, alkyl, halogen, -OR ⁸ , -SR ⁸ , -(NR R ⁹ ), -0S0 ₂ R ¹⁹ , a cyclic substituent, or a TAIL For all embodiments of the present mvention, preferably R ⁴ is not hydrogen In one embodiment of the mvention R ⁴ is halogen, -OR ⁸ , -SR ⁸ , -(NR ⁸ R ⁹ ), or -OS0 ₂ R ¹⁹ In another embodiment of the invenUon R ⁴ is an alkyl havmg 1 -6 carbons. In yet another embodiment of the mvention, R ⁴ is a TAIL

Where R and R taken in combmation form a fused 6-membered aromatic πng, embodiments of this mvention are quinolinium deπvatives according to the formula

where πng substituents R ¹ ' , R ¹² , R ¹³ , and R ¹⁴ may be the same or different, and are mdependently H, or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group havmg 1 -6 carbons, or a halogen, or -OR ⁸ , -SR ⁸ , -(NR ⁸ R ⁹ ), where R ⁸ and R ⁹ are as defined previously, or a cyclic substituent, as defined for R ⁵ , or a TAIL Preferred embodiment of the mvention are quinoliniums wherem m = 1 and p = 0 ("4-quιnolιnιums")

Typically, one or more of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁿ , R ¹² , R ¹³ and R ¹⁴ is a TAIL In one embodiment of the mvention, at least one of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ¹ ', R ¹² , R ¹³ and R ¹⁴ is required to be a TAIL Preferably.at least one of R ⁴ , R ⁶ , or R ¹² is TAIL, more preferably, R ⁴ is a TAIL When R ⁴ is a TAIL, LINK is preferably -NR ²⁰ - or -S- When TAIL is at any position other than R ⁴ or R ⁵ , LINK is preferably -O- or a smgle bond

In one embodiment of the invention, R ⁵ is a TAIL, and one of R ³ , R ⁴ , R ⁶ , R ⁷ , R ¹¹ , R ¹² , R ¹³ or R ¹⁴ is not hydrogen, preferably R ⁴ is not hydrogen

In another embodiment of the mvenUon, R ⁵ is not a cyclic substituent, and R ⁴ is not hydrogen Compounds wherem R ⁴ is not hydrogen possess significant advantages for stainmg nucleic acids In particular, dyes where R ⁴ is not hydrogen possess enhanced quantum yields relative to similar dyes wherem R ⁴ is H For this class of dyes, R ⁵ is preferably alkyl havmg 1 -6 carbons R ⁴ is typically -OR ⁸ , -SR ⁸ , -(NR ⁸ R ⁹ ), or R ⁴ is a TAIL, preferably R ⁴ is a TAIL In a specific embodiment of the invention, the dyes of the mvention are 4-pyndmιums or 4-quιnolιnιums, wherem R ⁵ is

an alkyl havmg 1 -6 carbons, and R ⁴ is not hydrogen

In yet another embodiment of the invention, R ⁵ is a cyclic substituent, and at least one of R ³ , R ⁴ , R ⁶ , R ⁷ , R ^{1 1} , R ¹² , R ¹³ or R ¹⁴ is a TAIL havmg a CAP that is -N ⁺ R^R^R ²³ ψ ^" , or a CAP that incorporates incorporates a 4- to 10- membered nng, preferably a 5- or 6-membered nng, that contains at least one nitrogen atom

In an additional preferred embodiment of the mvention, the second heterocyclic nng contains exactly two non- hydrogen substituents, one of which is a TAIL

Some of the dyes of the present invention that possess a TAIL moiety at R ⁴ exhibit particular utility for stainmg cells and microorganisms The utility of specific embodiments of the dyes of the present invention in stainmg cells and microorganisms is generally dependent on the chemical nature of the TAIL moiety, and the identity of the group present at R ⁵ For example, those compounds for which CAP is a quaternary ammomum salt are generally impermeant to living cells, with the exception of some yeast cells However, the permeability of those compounds for which CAP is a pπmary or secondary amine, and LINK is a secondary or tertiary amine, are related to the nature of R ⁵ (the N- substituent) Where R ⁵ is an aryl group, the compounds are generally permeant to all cells, living or dead, but the correspondmg compounds havmg an alkyl substituent at R ⁵ are generally impermeant to cellular membranes A similar relationship to the R ⁵ substituent is observed where TAIL is a nitrogen-containing heterocycle Where R ⁵ is an aryl group, the compounds are generally permeant to all cells, but when R ⁵ is an alkyl group, the dyes are generally permeant only to mammalian cells

Typically, dyes useful as impermeant cellular probes are those dyes havmg 2-3 positive charges, preferably 3 positive charges, more preferably havmg 2-3 positive charges where R ⁵ = alkyl Preferred dyes for permeant cellular probes are dyes wherem R ⁵ is alkyl having 1 -6 carbons, aryl or heteroaryl and CAP is -O-R ²¹ or -S-R ²¹ Dyes that are preferred for stainmg electrophoretic gels typically have CAP that is a dialkylammo group

A list of selected emodiments of the present mvention is presented m Tables 3, 4 and 5 While the table mcludes many preferred embodiments, the dyes shown m the Tables are not tended to be an exclusive list of die dyes of the present mvention Numerous modifications, substitutions, and alterations in substituents and dye structure are possible without departing from the spint and scope of the mvention

Table 3 : Specific examples of 4-quιnolιnιum dyes

Dye X R ⁴ R ⁵ R 12

211 O phenyl H

298 phenyl -OCH,

— N

308 phenyl -OCH,

— N -

309 phenyl -OCH,

314 O phenyl H

316 o phenyl H

--S"

342 o phenyl H

--N

-Nr"

345 o phenyl H

352 o phenyl H ι- /

365 phenyl H

-S ΛΛ

374 n-butyl phenyl

-oΛ fc

377 -CH, H

— N

Table 4: Specific examples of 4-pyridιmum dyes

Table 5: Specific examples of 2-quinolinium dyes

Synthesis

A useful synthetic route to the dyes of the present invention can be descnbed in three parts, following the natural breakdown m the descnption of the compounds In general, the synthesis of these dyes requires three precursors a benzazolium salt, a pyndinium (or quinolinium) salt (both of which have the appropnate chemical substituents, or can be converted to the appropπate substituents), and (where n = 1 or 2) a source for the methine spacer The combmation that enables these compounds to be useful stams for nucleic acids has not been descnbed previously, but the chemistry that is required to prepare and combme these precursors so as to yield any of the subject deπvatives is generally well- understood by one skilled m the art Although there are many possible vaπations that may yield an equivalent result, we provide herein some useful general methods for their synthesis and incorporation of chemical modifications

The pyndinium or quinolinium moiety

The strongly conjugated nng system of the compounds of the present mvention allows resonance stabilization of the smgle positive charge on the nng atoms to be distπbuted over the entire molecule In particular, the charge is stabilized by partial localization on each of the heterocyclic nitrogen atoms of the dye As the subject dye is drawn herein, the positive charge is formally localized on the benzazolium portion of the dye However, it is commonly understood that a comparable resonance structure can be drawn m which the positive charge is formally localized on the pyndimum portion of the dye Consequently, we will usually refer to this latter portion of the molecule as a pyndine, pyndimum, qumolme or quinolinium moiety, although in the resonance structure shown, it would formally be termed a dihydropyndine or dihydroqumolme

Compounds containmg the quinolinium moiety m this mvention differ from those that contam a smgle pyndimum nng only m the presence of an additional aromatic nng containing four carbon atoms that is fused at the R ⁶ and R ⁷ positions of the parent structure Except where reference is to a specific pyndine or pyndinium salt, it is understood that mention of pyndines or pyndimum salts encompasses benzopyndines and benzopyπdmium salts, which are formally called quino nes or quinolinium salts Mention of quinohnes and quinolinium salts refer only to structures containmg two fused aromatic rmgs

In the synthesis of the dyes of the mvention, the second heterocyclic precursor is usually a pyndimum salt that is already appropπately substituted (as m Examples 2, 9, and 11 ) Alternatively, substituents can be incorporated mto the pyndimum structure subsequent to attachment of the benzazolium portion of the dye (Example 5) One of the substituents, which may be incorporated before or after reaction with the benzazolium precursor is TAIL

Aside from the structural differences between pyndines and quinohnes, there exist two major structural distinctions within the family of dyes descnbed m the mvention, related to the pomt of attachment of the pyndimum moiety In one case (where m = 0 and p = 1 ) the position of attachment places the methine bndge adjacent to the nng nitrogen (2 -pyndines) In the more common case (where = 1 and p = 0) the position of the nitrogen atom is para to the pomt of attachment (4-pvndιnes)

Typically the required pyndimum salt precursor has the structure

and the qumolmium salt precursor has the general structure

with the substituents as defined previously At all times, the nng is a 6-membered pyndinium-based heterocycle

When n = 0, B is methyl, or B is chloro, bromo or iodo When n = 1 or 2, B is methyl Only when n = 1 or n = 2 is any part of B incorporated in the final compound

There are several general methods for the synthesis of deπvatives of pyndimum, mcludmg those deπvatives havmg substituents at any available position, mcludmg substitutions that are TAIL or that can be converted to TAIL before or after reaction with the benzazolium portion to form the dye core structure Substitutions at R ⁵ or at the position immediately adjacent to the nitrogen atom to which R ⁵ is attached (I e at R ⁴ when m = 1 and p = 0) are particularly important

Method 1 Alkylation of the nitrogen atom of an appropπately substituted qumolme with an alkylating agent such as a primary aliphatic halide, sulfate ester, sulfonate ester, epoxide or similar reagent directly yields a substituted qumolmium salt For example, treatment of a qumolme with 1 ,3-dιιodopropane and base, followed by heating with tπmethylamme, yields a TAIL substituent at R ⁵ (Example 18) If there is a TAIL substituent, or a group that can be converted to a TAIL substituent, at a position other than R ⁵ , then simple alkylatmg agents such as methyl iodide or dimethyl sulfate suffice to add the R ⁵ substituent, where R ⁵ is alkyl

Method 2 R ⁵ substituents that are aryl or heteroaryl are best incorporated by an Ullmann reaction of aniline or a substituted aniline or of a pyπdone or qumolone denvative In this method, a diaryl amine or aryl-heteroaryl amine (generally commercially available) is condensed with diketene and acid to yield a 4-methyl-N-arylquιnolone or a 4- methyl-N-heteroarylquinolone (as in Example 1 )

In the above formula, ARYL can be any aromatic or heteroaromatic nng system Further, R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , and substituents on ARYL may be a TAIL, or may be substituents that can be readily converted to a TAIL (Example 10)

The 4-methyl-2-quιnolone is then converted to the desired 4-methyl-2-substituted-qumolmιum salt by reaction with an organometal c reagent such as a Gngnard or organo thium reagent (Examples 9 and 10)

An R ⁴ substituent attached in this way may be aromatic or aliphatic and can be a TAIL or can be converted to TAIL (as m Example 13), provided that the nature of the substituent does not interfere with preparation of the required organometalhc reagent

Pyπdone and quinolone precursors may also be prepared by an Ullmann reaction of the appropπately substituted precursor if the nitrogen atom is hydrogen-substituted such as by the following reactions

While a vanety of 4-methyl-2-quιnolones are commercially available, desired deπvatives can be synthesized by reaction of aniline or a substituted-aniline with an acetoacetate or acetoacetate equivalent reagent such as diketene

diketene

Pyndone and qumolone intermediates containmg a non-hydrogen group at R ⁵ are particularly important precursors to a wide vanety of other pyndimum and qumolmium salts that are substituted at R ⁴ In particular, a salt is formed by treatment of the appropnate pyndone or qumolone with a strong chlormatmg agent such as PC1 ₅ , POCl ₃ or SOCl ₂ , for mstance m the reaction below (Example 2) Smularly, a sulfonate can be substituted at R ⁴ by treatmg the pyndone or qumolone with the appropπate sulfonic acid anhydnde

Halogen displacement

The reactivity of the 2-halogenated pyndimum or qumolmium intermediate offers a vanety of synthetic methods for attachment of vanous substituents at the 2-posιtιon, mcludmg TAILs and TAIL precursors However, the reactivity of the 2-halo denvatives is preserved even after conjugation with the benzazolium precursor, enabl g conversion of the resulting dye m which R ⁴ is halogen mto the appropnate alkoxy, ammo and thiolate analogs, as descnbed for the pyndimum and qumolmium precursors Of particular utility for the dyes of the present mvention is the displacement of a 2-chloro substituent by amines (yielding TAIL or TAIL precursors where LINK is -NR ²⁰ -), thiols (yielding TAIL or TAIL precursors where LINK is -S-) or alcohols (yielding TAIL or TAIL precursors where LINK is - 0-) The displacement of chlonde by amines is descnbed m Example 5, and the displacement of chlonde by thiols is descnbed m Example 7

Additionally, the 2-oxo group of pyndone or qumolone precursors can be chemically reduced to deπvatives in which R ⁴ is H usmg a vanety of reagents mcludmg DD3AL-H (diisobutylaluminum hydnde)

IΔJ

As descnbed earlier, TAIL is composed of three parts LINK, SPACER and CAP If a TAIL is present as R ⁵ , then LINK is constrained to be a smgle bond, eliminating the potential of N-S, N-O or N-N bonds m TAIL The chemical composition of SPACER is determmed by the chemistry required to attach the heteroatom m CAP with the dye core structure via LINK

As descnbed above, those dyes of the present invention that are 4-pyndιnιums or 4-quιnolιnιums wherein R ⁴ is a TAIL are most conveniently synthesized from the 2-halopyndιnιum or 2-haloquιnolιnιum precursor either before or after condensation with the benzazolium portion of the dye by a nucleophilic displacement reaction of the halogen by a thiol, alkoxide, or a primary or secondary amine

CAP ma> be incorporated directly mto TAIL before or after condensation of the pyndimum or qumolmium salt with the benzazolium salt, or CAP may be added or further modified at a later stage in the synthesis For mstance, when CAP is a cyclic or non-cyclic primary, secondary or tertiary amine, CAP can be alkylated to a quaternary ammomum (Examples 6, 7 and 8) This reaction can be used to mcrease the polaπty of the dye and to thus restnct its penetration through the membrane of living cells, and to additionally mcrease the dye's affinity for nucleic acids

Precursors to TAIL mclude carboxylic acids, halides, alcohols and thiols Each of these reactive groups can be used to attach a heteroatom containing moiety (I e , CAP) to the dye core structure, generally through the formation of amides (Example 15 and 16), ethers or thioethers, which are incorporated mto SPACER before (Example 15) or after (Example 16) attachment of SPACER to the dye core structure

Condensation of the dve

The benzazolium precursors are prepared and condensed with the pyndimum or qumolmium salts according to the synthetic procedures outlmed in U S Patent No 5,436,134 to Haugland et al (1995) The specific method of conjugation and reagents used result m a methine, tπmethine or pentamethine bndge between the two πng systems

Method of Use

The use of the invention compπses combining a dye of the present invention with a sample that contains or is thought to contam a nucleic acid polymer, incubating the mixture of dye and sample for a time sufficient for the dye to combine with nucleic acid polymers m the sample to form one or more dye-nucleic acid complexes havmg a detectable fluorescent signal The charactenstics of the dye-nucleic acid complex, mcludmg the presence, location, intensity, excitation and emission spectra, fluorescence polarization, fluorescence lifetime, and other physical properties of the fluorescent signal can be used to detect, differentiate, sort, quantitate, and/or analyze aspects or portions of the sample The dyes of the invention are optionally used m conjunction with one or more additional reagents (preferably detectably different fluorescent reagents), mcludmg dyes of the same class havmg different spectral properties

Stainmg Solution

Typically, the subject dye is prepared for use by dissolving the dye m a staining solution, preferably an aqueous or aqueous-miscible solution that is compatible with the sample and the tended use For biological samples, where minimal perturbation of cell moφhology or physiology is desired, the stammg solution is selected accordmgly The dye is typically dissolved directly in an aqueous solvent such as water or a buffer solution, such as buffered saline, or is dissolved m a water-miscible organic solvent such as dimethylsulfoxide (DMSO), dimethylformamide (DMF), or a lower alcohol such as methanol or ethanol, at a concentration of greater than about 100-tιmes that used in the stammg solution, then diluted one or more times with an aqueous solvent such as water or buffer, such that the dye is present m an effective amount

An effective amount of dye is the amount sufficient to give a detectable fluorescence response m combmation with nucleic acids The dye concentration in the solution must be sufficient both to contact the nucleic acids in the sample and to combine with the nucleic acids in an amount sufficient to give a signal, while minimizmg background fluorescence Typically staining solutions for cellular samples have a dye concentration greater than 0 1 nM and less than 50 μM, more typically greater than 1 nM and less than 10 μM, preferably between 0 5 and 5 μM In general, lower concentrations of dyes are required for eukaryotes than for prokaryotes, and for dyes with higher sensitivity Stainmg solutions for electrophoretic gels typically have a dye concentration of greater than 0 1 μM and less than 10 μM, more typically about 0 5-2 μM, the same holds true where the dye is added to the gel (pre-cast) before bemg combmed with nucleic acids Stainmg solutions for detection and quantitation of free nucleic acids m solution typically have a concentration of 0 1 μM-2 μM The optimal concentration and composition of the stainmg solution is determmed by the nature of the sample (mcludmg physical, biological, biochemical and physiological properties), the nature of the dye- sample interaction (including the transport rate of the dye to the site of the nucleic acids), and the nature of the analysis bemg performed, and can be determmed according to standard procedures such as those descnbed m examples below

Sample Types

The dve is combined with a sample that contains or is thought to contam a nucleic acid The presence of the

nucleic acid m the sample may be due to natural biological processes, or the result of a successful or unsuccessful synthesis or experimental methodology, undesirable contamination, or a disease state The nucleic acid may be endogenous or introduced as foreign matenal, such as by infection, transfection, or therapeutic treatment

The nucleic acid m the sample is RNA or DNA, or a mixture or a hybnd thereof Any DNA is optionally smgle-, double-, tπple-, or quadruple-stranded DNA, any RNA is optionally smgle stranded ("ss") or double stranded ("ds") The nucleic acid may be a natural polymer (biological in oπg ) or a synthetic polymer (modified or prepared artificially) The nucleic acid polymer (preferably containmg at least 8 bases or base pairs) may be present as nucleic acid fragments, oligonucleotides, or larger nucleic acid polymers with secondary or tertiary structure The nucleic acid is optionally present m a condensed phase, such as a chromosome (Examples 26, 27) The nucleic acid polymer optionally contams one or more modified bases or links or contams labels that are non-covalently or covalently attached For example, the modified base can be a naturally occurring modified base such as ψ (pseudoundine) m tRNA, 5- methylcytosine, 6-methylammopunne, 6-dιmethylammopunne, 1 -methylguanine, 2-methylamιno-6-hydroxypuπne, 2- dιmethylammo-6-hydroxypunne, or other known minor bases (see, e g Davidson, THE BIOCHEMISTRY OF THE NUCLEIC ACIDS (1976)) or is synthetically altered to contam an unusual linker such as moφholine deπvatized phosphates (Anti Virals, Inc , Corval s, OR), or peptide nucleic acids such as N-(2-amιnoethyl)glycιne units (Wittung, et al , Nature 368, 561 (1994)) or contam a simple reactive functional group (<10 carbons) that is an aliphatic amine, carboxylic acid, alcohol, thiol or hydrazine, or contam a fluorescent label or other hapten, where the label is oπginally attached on the nucleotide or on the 3' or 5' end of the polymer, or ligands non-covalently attached to the nucleic acids (e g Examples 28, 29) Typical modified bases mclude mosme, bromodeoxyuπdine and lododeoxyundine, and bases labeled with haptens such as biotm, digoxigemn, or 2,4-dmιtrophenyl, or with fluorophores The presence of such labels does not significantly interfere with DNA amplification or with subsequent analysis on gels or in solution (Example 33, Figure 2)

The sample that contams the nucleic acid is optionally a biological structure (I e an organism or a discrete unit of an organism), or a solution (mcludmg solutions that contam biological structures), or a solid or semi-solid matenal Consequently, the nucleic acid used to practice the mvention is optionally free m solution, immobilized m or on a solid or semi -solid matenal, extracted from a biological structure (e g from lysed cells, tissues, organisms or organelles), or remains enclosed within a biological structure The biological structure that encloses the nucleic acid is optionally a cell or tissue, for example where the nucleic acid is present m a cell or interstitial space as a prokaryote or eukaryote microorgamsm, or as a virus, viroid, chromosome or organelle Alternatively, the biological structure is not enclosed m a tissue or cell, and is present either as a virus or as a microorgamsm or other cell, or is present as a cellular component removed from its parent cell (e g a plasmid or chromosome, or a mitochondπon or nucleus or other organelle)

Typically, the sample is a cell or is an aqueous or aqueous miscible solution that is obtamed directly from a liquid source or as a wash from a solid matenal (organic or inorganic) or a growth medium m which cells have been introduced for culturmg or a buffer solution in which nucleic acids or biological structures have been placed for evaluation Alternatively, the sample is a solid, optionally a smear or scrape or a retentate removed from a liquid or vapor by filtration In one aspect of the mvention, the sample is obtamed from a biological fluid, such as urine,

cerebrospinal fluid, blood, lymph fluids, tissue homogenate, interstitial fluid, cell extracts, mucus, saliva, sputum, stool, physiological secretions or other similar fluids Alternatively, the sample is obtamed from an environmental source such as soil, water, or air, or from an mdustnal source such as taken from a waste stream, a water source, a supply l e, or a production lot Industrial sources also mclude fermentation media, such as from a biological reactor or food fermentation process such as brewmg, or foodstuffs, such as meat, gram, produce, or dairy products (Example 42)

The sample solution can vary from one of purified or synthetic nucleic acids such as ohgonucleotides to crude mixtures such as cell extracts or homogenates or other biological fluids, or dilute solutions from biological, mdustnal, or environmental sources In some cases it is desirable to separate the nucleic acids from a mixture of biomolecules or flmds m the solution pnor to combmation with the dye Numerous techniques exist for separation and purification of nucleic acids, mcludmg such means as chromatographic techniques and electrophoretic techniques, using a vanety of supports or solutions or m a flowing stream, or usmg nuclease degradation

Cell types for which the dye is an effective nucleic acid stain mclude cells with or without nuclei, mcludmg but not limited to, eukaryotes, such as plant and animal cells (particularly vertebrate cells), mcludmg pollen and gamete cells, prokaryotes, particularly bactena, mcludmg both Gram-negative and Gram-positive bactena, as well as yeast and other fungi, and spores The cells are optionally smgle cells, mcludmg microorgamsms, or multiple cells associated with other cells m two or three dimensional layers, mcludmg multicellular organisms, embryos, tissues, biopsies, filaments, biofilms, etc The cells are viable or dead cells or a mixture thereof Alternatively, the cells are blebbmg or undergoing apoptosis or in a cycle of growth or cell division The dyes are not equally effective m stammg all cell types and certain dyes are generally more permeant than others Live cells are less permeable to the dyes than dead cells, and prokaryotes are less permeable than eukaryotes (see Table 6) Some of the dyes are generally not permeant to live cells with intact membranes, other dyes are generally permeant to eukaryotes but not to prokaryotes, still other dyes are only permeant to cells m which the cell membrane mtegnty has been disrupted (e g some dead cells) The relative permeability of the cell membrane to the dyes is determmed empincally, e g by companson with stammg profiles or stainmg patterns of killed cells The dye with the desired degree of permeability, and a high absorbance and quantum yield when bound to nucleic acids, is selected to be combmed with the sample

Furthermore, the differential permeability of bacteπal and higher eukaryotic dells to some dyes allows selective stainmg of live mammalian cells with little or no stainmg of live bactena A dye selected to be permeant to bactena can be used m combmation with a dye that is only permeant to eukaryotes to differentiate bactena m the presence of eukaryotes Dead bactena with compromised membranes, such as those m the phagovacuoles of active macrophages or neutrophils, may be rendered permeable to the dyes that are otherwise only permeant to eukaryotes, as a result of toxic agents produced by the phagocvtic cells (Example 44)

Table 6 Permeability of selected dyes of the invention

19

KEY

Scale of bnghtness, 1 = dim, 4 = bnght

Killed - 70% Isopropanol killed, Dead - 3 7% formaldehyde killed, Fixed/Permeabilized - Dead, then acetone fixed

DNA - Stams DNA spot on Yeast

Mitoch - Stains m tochondna, either spotty or whole Cyto Bkg - Cytoplasmic background, 0 = no background, 4 = high background

Formation of Dye-Nucleic Acid Complex

The sample is combmed with the stainmg solution by any means that facilitates contact between the dye and the nucleic acid, typically through simple mixing Where the sample is a solution, the stainmg solution is added to the sample solution directly or m a liquid separation medium such as an electrophoretic liquid, sievmg matnx or running buffer, or m a sedimentation (e g sucrose) or buoyant density gradient (e g containing CsCl), or on an mert matnx such as a blot or gel, a testing strip, or any other solid or semi-solid support Suitable supports also mclude, but are not limited to, polymeπc microparticles (mcludmg paramagnetic microparticles), polyacrylamide and agarose gels (both denaturing and non-denaturing), nitrocellulose filters, computer chips (such as silicon chips for photolithography), natural and synthetic membranes, liposomes and algmate hydrogels, and glass (mcludmg optical filters), and other silica- based and plastic support The dye is optionally combmed with the nucleic acid solution pπor to undergoing gel or capillary electrophoresis, gradient centnfugation, or other separation step, during separation, or after the nucleic acids undergo separation Alternatively, the dye is combmed with an mert matnx or solution a capillary pnor to addition of the nucleic acid solution, as m pre-cast gels, capillary electrophoresis or preformed density or sedimentation gradients

Where the nucleic acids are enclosed in a biological structure, the sample is typically incubated with the dye but any other technique that is suitable for transporting the dye mto the biological structure can be used Some cells actively transport the dyes across cell membranes (e g endocytosis or mgestion by an organism or other uptake mechanism) regardless of their cell membrane permeability Suitable artificial means for transporting the dyes (or pre- formed dye-nucleic acid complexes) across cell membranes mclude, but are not limited to, action of chemical agents such as detergents, enzymes or adenosme tnphosphate, receptor- or transport protein-mediated uptake, liposomes or algmate hydrogels, phagocytosis or other types of mgestion, pore-forming proteins, microinjection, electroporation, hypoosmotic shock, or minimal physical disruption such as scrape loading, patch clamp methods, or bombardment with solid particles coated with or in the presence of the dyes Preferably, where mtact structures are desired, the methods for staining cause minimal disruption of the viability of the cell and integπty of cell or intracellular membranes

Alternatively, the cells are fixed and treated with routine histochemical or cytochemical procedures, particularly where pathogenic organisms are suspected to be present The cells are typically fixed immediately after stammg with an aldehyde fixative that keeps the dye m the cells The fixation of cells produces extensive cross-linking within the cellular membranes, so that membranes that were mtact at the time of fixation remain impermeable to the dead cell stam Pathogenic bactena can therefore be assayed for viability after fixation, reducing the nsk of exposure Typically cells are fixed using a pH-buffered fixative, such as 3 7% formaldehyde or 4% glutaraldehyde Upon fixation, the sample cells are preferably washed to remove excess fixative, then stamed In some cases, live or dead cells are fixed pnor to stainmg without substantially mcreasmg cell membrane permeability of previously live cells so that only cells that were already dead pnor to fixation stam with the cell-impermeant dye

The sample is combined with the dye for a time sufficient to form the fluorescent nucleic acid-dye complex, preferably the minimum time required to give a high signal-to-background ratio The optimal time is usually the minimum time required for the dye, m the concentration bemg used, to achieve the highest target-specific signal while avoidmg degradation of the sample over time and minimizing all other fluorescent signals due to the dye For example,

where the dye is chosen to be selective for a particular nucleic acid polymer or type of cell, the optimal time is usually the minimum time required to achieve the highest signal on that polymer or type of cell, with little to no signal from other nucleic acids or other cell types

Preferably, the dye is combmed with the sample at a temperature optimal for biological activity of the nucleic acids within the operating parameters of the dyes (usually between 5 °C and 50 °C, with reduced stability of the dyes at higher temperatures) For in vitro assays, the dye is typically combmed with the sample at about room temperature (23 °C) At room temperature, detectable fluorescence m a solution of nucleic acids is essentially instantaneous depending on the sensitivity of the instrumentation that is used, fluorescence solutions is generally visible by eye within 5 seconds after the dye is added, and is generally measurable within 2 to 5 mmutes, although reachmg equilibnum stainmg may take longer Gel stainmg at room temperature usually takes from 5 mmutes to 2 hours depending on the thickness of the gel and the percentage of agarose or polyacrylamide, as well as the degree of cross-linking Typically, post- stained mimgels stam to equilibnum m 20-30 mmutes For cells and other biological structures, transport of dyes across membranes is required whether the membranes are mtact or disrupted For preferred embodiments, visibly detectable fluorescence is obtained at room temperature within 15-20 mmutes of incubation with cells, commonly within about 5 mmutes Some embodiments give detectable fluorescence inside cells m less than about 2 mmutes Lymphocytes loaded with 5 μM dye solutions give a fluorescence response m less than 5 seconds This property is useful for observing nuclear structure and rearrangement, for example such as occurs during mitosis or apoptosis

Fluorescence of the Dye-Nucleic Acid Complex

The nucleic acid-dye complex formed duπng the stainmg of the sample with a dye of the present invention compnses a nucleic acid polymer non-covalently bound to one or more molecules of dye The combmation of dye and nucleic acid results m a fluorescent signal that is significantly enhanced over the fluorescence of the dye alone Where the fluorescence of the dye-nucleic acid complex decreases at pH lower than 6 5 or greater than 8, it is typically restored by returning to moderate pH

The quantum yield of unbound dye is typically <0 01 , usually <0002, and frequently <0 001 , which would yield a maximum enhancement of > 1 OOx, >500x, and > 1 OOOx respectively For most applications, dyes are selected to give a quantum yield greater than about 0 3, preferably greater than 0 6, when bound to nucleic acid The level of fluorescence enhancement of the bound dye is generally about 100-1000 fold greater than that of unbound dye, typically greater than about 200-fold, such that the dyes have a readily detectable mcrease in quantum yield upon bmdmg to nucleic acids More typically, the fluorescence enhancement is greater than 300-fold, preferably greater than 1000 fold The molar absoφtivity (extinction coefficient) at the longest wavelength absoφtion peak of the dyes is typically > 50,000 and frequently > 60,000 for the dyes where n = 0, for dyes where n = 1 or 2, the molar absoφtivity is typically greater than 90,000 Dyes with high extinction coefficients at the excitation wavelength are preferred for the highest sensitivity

A useful level of quantum yield in combmation with other attπbutes of the subiect dyes, including selectivity

NOT TO BE CONSIDERED FOR INTERNATIONAL PUBLICATION

Table 7: Quantum Yields of Indicated Nucleic AcidDye Complexes

Fluorescence properties of dyes. Quantum yields of selected dyes bound to double-stranded calf thvmus DNA (ds DNA), E coli nbosomal RNA, single-stranded Ml 3 phage DNA (ss DNA) and oligonucleotides (a synthetic 24 mer)

Fluorescence excitation and emission maxima on double-stranded DNA are mdicated, units are nm

The fluorescence of the complex is detected qualitatively or quantitatively by detection of the resultant light emission at a wavelength of greater than about 450 nm, preferably greater than about 480 nm, more preferably at greater than about 500 nm Dyes havmg a qumolmium πng system usually absorb and emit at longer wavelength maxima than smularly substituted dyes havmg a pyndimum nng system The emission is detected by means that mclude visual inspection, CCD cameras, video cameras, photographic film, or the use of current instrumentation such as laser scannmg devices, fluorometers, photodiodes, quantum counters, plate readers, epifluorescence microscopes, scannmg microscopes, confocal microscopes, flow cytometers, capillary electrophoresis detectors, or by means for amplifymg the signal such as a photomultipher tube Many such instruments are capable of utilizing the fluorescent signal to sort and quantitate cells or quantitate the nucleic acids, e g image analysis systems or flow cytometers (Examples 47, 48, 49) Dyes can be selected to have emission bands that match commercially available filter sets such as that for fluorescem or for detecting multiple fluorophores with several excitation and emission bands

Use of Complex

Once the dye-nucleic acid complex is formed, its presence may be detected and used as an indicator of the presence, location, or type of nucleic acids in the sample, or as a basis for sorting cells, or as a key to characterizing the sample or cells in the sample (Tables 6-9 and 12, Example 41 ) Such characterization may be enhanced by the use of additional reagents, mcludmg fluorescent reagents Attachment of covalent labels to the polymers used to form the dye- nucleic acid complex does not prevent subsequent formation of the fluorescent complex (Figure 2, Example 33) In addition to use m qualitative analysis, the nucleic acids in a sample can also be quantified by compaπson with known relationships between the fluorescence of the nucleic acid-dye complex and concentration of nucleic acids in the sample (Examples 21, 22, 38, 48, Figures 3, 4, 10)

The dyes of the mvention give a strong fluorescent signal with small nucleic acid polymers (as few as 8 bases or base pairs with some embodiments) even with very small amounts of nucleic acids Us g a fluorescence microscope, a smgle nucleic acid molecule can be detected (Example 37) Nucleic acid content from as few as 5 mammalian cells can be detected in cell extracts (Example 38) As little as 25 picograms of ds DNA/mL of solution is detected in a fluorometer (Example 21 ) In conjunction with an ultraviolet transilluminator, it is possible to detect as little as 10 picograms of ds DNA per band in an electrophoretic gel (Example 19), some dyes give such a bπght signal even with illummation by ordinary fluorescent room lights, that as little as 1 ng DNA per band is detected When used for pre- or post-staining of electrophoresis gels, the high sensitivity of the dyes of the present mvention allows the detection of previously unmeasurable amounts of nucleic acids using inexpensive instrumentation (e g UV trans- and epi-illuπunators) without requiπng destaining (see Table 8)

Table 8 Sensitivity of nucleic acid detection m electrophoretic gels

For each test, a dilution seπes of λcI857 DNA cut with Hind III restnction endonuclease or a dilution senes off coli nbosomal RNA was electrophoresed in 10 cm x 10 cm x 0 4 cm 1 % agarose gels Gels were poststamed with a 1 μM solution of each dye m TBE buffer for 20 mmutes and photographed through a Wratten 15 gelatin filter, with Polaroid black and white pnnt film, us g 254 nm epi-illumination or 300 nm transillummation as mdicated Numbers mdicate the amount of nucleic acid in the lowest intensity band that was visible in the Polaroid photograph, bands were 3 5 mm

Alternatively, the presence or location of nucleic acids, stained as above, can in turn be used to mdicate the presence or location of orgamsms, cells, or organelles containmg the nucleic acids, where the presence or location of the fluorescent signal corresponds to the presence or location of the biological structure (e g stained cells or organelles) Infective agents such as bactena, mycoplasma, mycobactena, viruses and parasitic microorgamsms, as well as other cells, can be stained and detected inside of eukaryote cells, although the fluorescent signal generated by an individual virus particle is below the resolution level of standard detection instrumentation In a further embodiment of the mvention the fluorescent signal resulting from formation of the dye-nucleic acid complex is used as a basis for sorting cells, for example sorting stained cells from unstained cells or sorting cells with one set of spectral properties from cells with another set of spectral properties (Examples 45, 47, Figures 8, 9)

In addition to detection of the presence or location of nucleic acids as well as their enclosing structures, the stammg profile that results from the formation of the dye-nucleic acid complex is indicative of one or more charactenstics of the sample By stainmg profile is meant the shape, location, distnbution, spectral properties of the profile of fluorescent signals resultmg from excitation of the fluorescent dye-nucleic acid complexes The sample can be characterized simply by stainmg the sample and detecting the stainmg profile that is indicative of a characteπstic of the sample More effective characterization is achieved by utilizing a dye that is selective for a certain charactenstic of the sample or by utilizing an additional reagent (see below), where the additional reagent is selective for the same charactenstic to a greater or lesser extent or where the additional reagent is selective for a different charactenstic of the same sample

In one embodunent of the mvention, mtegnty of cell mebranes is determmed by stainmg cells as above for a time penod and dye concentration sufficient to give a detectable fluorescent signal m cells with compromised membranes Where the dye selected is impermeant to cells with mtact membranes, formation of the fluorescent dye- nucleic acid complex inside the cell is indicative that the mtegnty of the cell membrane is disrupted and the lack of fluorescent dye-nucleic acid complexes mside the cell is indicative that the cell is mtact or viable The impermeant dye is optionally used in conjunction with a counterstam that gives a detectably different signal and is indicative of metabohcally active cells or, m combmation with the impermeant dye, is indicative of cells with mtact membranes Alternatively, the more permeant dyes of the mvention are used to stam both cells with mtact membranes and cells with disrupted membranes, m conjunction with a counterstam that gives a detectably different signal m cells with disrupted membranes, allowmg the differentiation of viable cells from dead cells The counterstam that gives a detectably different signal m cells with disrupted membranes is optionally an impermeant dye of the mvention or another reagent that indicates loss of mtegnty of the cell membrane or lack of metabolic activity of the dead cells When the cells are stained with a concentration of dye that is known to stam live bactena, the relative reduction of a fluorescence intensity can be used to distinguish quiescent bactena, which are not actively expressmg proteins, from metabohcally active bactena (Figures 8A & 8B, Example 45)

Table 9 Stainm Pattern of Selected D es

S YTO 14 and Hoechst 33342 are available commercially from Molecular Probes, Inc The designation "mitochondna" indicates staimng of the entire mitochondπon, the designation "mitochondπal nucleoids" indicates punctate staining within the mitochondnon, thought to be labeling of the mitochondπal DNA The designation "n d " mdicat dye/cell type combinations that were not tested

In a further embodunent of the mvention, the shape and distnbution of the staimng profile of dye-nucleic acid complexes is indicative of the type of cell or biological structure that contams the stained nucleic acids Cells may be discriminated by eye based on the visual fluorescent signal or be discriminated by instrumentation as descnbed above, based on the spectral properties of the fluorescent signal For example, dyes that are non-selective for staimng nucleic acids m intracellular organelles can be used to identify cells that have an abundance or lack of such organelles, or the presence of micronuclei and other abnormal subparticles containing nucleic acids and charactenstic of abnormal or diseased cells A sample may be characterized as containing blebbmg cells or nuclei based on the visible stammg profile Dyes that are selective for the nucleic acids m a particular organelle (e g m nucleus, mitochondna, mtiochondnal nucleoids), even in the presence of limited stammg of nucleic acids m the cytoplasm or other organelles, can be used to characterize cells as containmg or lackmg such organelles based on the mtensity as well as the location of the signal, allowmg the use of instrumentation to characterize the sample Typically the staimng profile used to characterize the sample is indicative of the presence, shape, or location of organelles or of cells, where the cells are located in a biological fluid, m a tissue, or m other cells Dyes that give a distnbution of fluorescence mtensity signals that reflects the distnbution of DNA content of a cell population can be used for cell cycle analysis

In another embodunent of the invention, the staimng profile results from the formation of the dye-nucleic acid complex in an electrophoretic gel, or sedimentation or centrifugation gradient In addition to indicating the presence of nucleic acids m the gel, the staimng profile is indicative of one or more charactenstics of the nucleic acid solution applied to the gel The number of bands and/or the mtensity of the signal per band of the stai ng profile, for example, is indicative of the punty or homogeneity of the nucleic acid solution Band tightness and degree of smearing is indicative of the mtegnty of the nucleic acid polymers in the solution The size, conformation, and composition of the polymers, are indicated by the relative mobility of the polymer through the gel (Examples 34, 35), which can be used to detect changes caused by interaction of analytes with the nucleic acid polymer such as protein bmdmg or enzymatic activity Preferred embodiments of the dyes have low intrinsic fluorescence so there is no need to destain gels to remove free dye Furthermore, the fluorescence of the dye-nucleic acid complex is not quenched by denaturants such as urea and formaldehyde, eliminating the need for their removal from the gels pnor to stainmg

In yet another embodiment of the mvention, the stammg profile is indicative of the presence or predominance of a type of nucleic acid that is used to characterize the sample In one embodiment of the mvention, the dye is chosen to be more selective for AT or GC nch polymers, such that stainmg profile is indicative of the relative proportion of these bases (Example 41 , Table 12) In another embodiment of the mvention, the spectral properties of the nucleic acid-dye complex vary depending on the secondary structure of the nucleic acid present m the complex Typically, the spectral properties will vary in fluorescence enhancement, fluorescence polarization, fluorescence lifetime, excitation wavelength or emission wavelength, preferably emission wavelength A compaπson of the fluorescence response of a sample of unknown nucleic acids with that of a stained nucleic acid of known secondary structure allows the secondary structure of the unknown nucleic acids to be determined, and the amount of nucleic acids m the sample to be quantified In this manner, RNA and single-stranded DNA can be differentiated from double-stranded DNA (Example 40) Where nuclease is added to the nucleic acid polymers m solution or in fixed cells to digest the RNA or DNA pπor to combining with the dye, the fluorescent signal from the dve-nucleic acid complex can be used to discriminate the nucleic

acid polymer that was not digested m the presence of the nuclease from undigested polymers (Example 39)

This same property of sensitivity to secondary structure by monomethine dyes can be used to quantitate ds nucleic acids the presence of ss nucleic acids Samples containing both ds and ss DNA or RNA yield emission maxima m both the green and longer wavelength regions at high dye base ratios Meaningful information about the amounts of ss and ds nucleic acids m solution can be gathered by a direct companson of the spectra of the low dye ratio sample and high dye ratio sample For example, where a nucleic acid solution such as purified o gonucleotides, DNA amplification reactions, a cDNA synthesis, plasmid preparation, or cell extraction is stained with a high dye concentration (I e greater than or equal to the concentration of nucleic acid bases), the fluorescent signal that results from complexes formed by ss nucleic acids is red-shifted from the fluorescent signal formed by ds nucleic acids Where the dye is selected to give a high quantum yield with ds nucleic acids and the quantum yield of the red-shifted fluorescent signal is minimal, the quantum yield of the stronger signal can be used to quantitate the amount of ds nucleic acid in the sample, even in the presence of ss nucleic acids (Example 40, Figure 6)

The nucleic acids for this and other applications are quantitated by companson of the detectable fluorescent signal from the dye-nucleic acid complex, with a fluorescent standard charactenstic of a given amount of nucleic acid (Examples 21 , 22, 38, 39, 48) The reference value of fluorescence used for companson with the fluorescence resulting from the formation of the dye-nucleic acid complex is determmed from a sample of known nucleic acid content or from a distnbution of nucleic acid content in a given population of cells over time Where one type of nucleic acid m a sample is selectively digested to completion, the fluorescent signal can be used to quantitate the polymer remaining after digestion (Example 39) Alternatively, pπor to being stained, a solution of nucleic acid polymers is separated mto discrete fractions usmg standard separation techniques and the amount of nucleic acid present m each fraction is quantitated usmg the mtensity of the fluorescent signal that corresponds to that portion The solution may be purified synthetic or natural nucleic acids or crude mixtures of cell extracts or tissue homogenates Where aliquots from a smgle sample are taken over time, and the nucleic acid content of each aliquot is quantitated, the rate of cell or nucleic acid proliferation is readily determmed from the change in the corresponding fluorescence over tune (Example 38), or from the change m cell distnbution over cell cycle compartments (Examples 48 and 49)

In another aspect of the mvention, the dye-nucleic acid complex is used as a fluorescent tracer or as probe for the presence of an analyte In one aspect of the mvention, the dye-nucleic acid complex is used as a size or mobility standard, such as m electrophoresis or flow cytometry Alternatively, the fluorescent signal that results from the interaction of the dye with nucleic acid polymers can be used to detect or quantitate the activity or presence of other molecules that mteract with nucleic acids The nucleic acid polymers used to form the dye-nucleic acid complex are optionally attached to a solid or semi-solid support, or free m solution, or enclosed m a biological structure Such molecules mclude drugs, other dyes, protems such as histones or ds or ss DNA or RNA bmdmg proteins, or enzymes such as endonucleases or topoiso erases In one aspect of the mvention, a dye havmg a bmdmg affinity for nucleic acid greater than that of the analvte bemg assayed displaces the analyte or prevents the interaction of the analyte with the nucleic acid polymer For example, DNA templates that are heavily bound with a high affinity dye such as dye 1 1 14 (l e at ratios of greater than 3 bp dye molecule m the stainmg solution) are protected from DNase I activity Typically

the dyes havmg a dissociation constant less than 10 ^"6 M, more typically less than 10" ⁸ M, are effective to displace analytes that mteract with nucleic acids Dye affinity is determmed by measuring the fluorescence of the dye-nucleic acid complex, fitting the resultmg data to an equilibnum equation and solvmg for the association constant In another aspect of the mvention, dyes havmg a bmdmg affinity that is less than that of the analyte bemg assayed are displaced from the dye-nucleic acid complex by the presence of the analyte, with the resultant loss of fluorescence For example, lower affinity dye molecules prebound to double-stranded DNA are displaced by histones

In one embodiment, the complex is used as an indicator of enzymatic activity, that is, as a substrate for nucleases, topoisomerases, gyrases, and other enzymes that mteract with nucleic acids (Example 24) Some embodiments of the dyes inhibit non-specific nuclease activity but not restnction endonuclease activity at certain dye base pair ratios Alternatively, the complex is used to quantitate the abundance of protems (such as histones) that bmd nucleic acids, or of DNA bmdmg drugs (such as distamycin, sper ine, actinomycin, mithramycin, chromomycin) The fluorescent complex is combmed with the sample thought to contam the analyte and the resultant mcrease or decrease m fluorescent signal qualitatively or quantitatively mdicates the presence of the analyte

Additional Reagents

The dyes of the mvention can be used in conjunction with one or more additional reagents that are separately detectable The additional reagents may be separately detectable if they are used separately, e g used to stam different aliquots of the same sample (e g Example 39) or if they stam different parts or components of a sample (e g Example

42), regardless whether the signal of the additional reagents is detectably different from the fluorescent signal of the dye- nucleic acid complex Alternatively, the dye of the mvention is selected to give a detectable response that is different from that of other reagents desired to be used m combmation with the subject dyes Preferably the additional reagent or reagents are fluorescent and have different spectral properties from those of the dye-nucleic acid complex For example, dyes that form complexes that permit excitation beyond 600 nm can be used in combmation with commonly used fluorescent antibodies such as those labelled with fluorescein lsothiocyanate or phycoerythrm

Any fluorescence detection system can be used to detect differences m spectral properties between dyes, with differing levels of sensitivity Such differences mclude, but are not limited to, differences m excitation and/or emission maxima, m fluorescence lifetimes, m fluorescence emission mtensity at the same or different excitation wavelengths, absoφtivity, m fluorescence polarization, m fluorescence enhancement m combmation with target matenals, or combmations thereof The detectably different dye is optionally one of the dyes of the mvention havmg different spectral properties and different selectivity In one aspect of the mvention, the dye-nucleic acid complex and the additional detection reagents have the same or overlappmg excitation spectra, but possess visibly different emission spectra, generally havmg emission maxima separated by >10 nm, preferably >20 nm, more preferably >50 nm Alternatively, the additional reagent(s) are simultaneously or sequentially excited at a wavelength that is different from that used to excite the sub _j ect dve-nucleic acid complex (e g Example 44) In yet another alternative, one or more additional reagents are used to quench or partially quench the fluorescence of the dye-nucleic acid complex, such as by adding a second reagent to improve the selectivity for a particular nucleic acid or the AT/GC selectivity

Improving on the procedure of Saitoh, et al , CELL 76, 609 (1994) for banding on metaphase chromosomes, green fluorescent dyes of the mvention bmd essentially nonselectively along the entire chromosome and are quenched by Methyl Green selectively bmdmg m regions with high AT content The result is a seπes of fluorescent green bands separated by dimmer high AT regions that are charactenstic of particular chromosomes (Example 27) This enables karyotype analysis and structural analysis, mcludmg the identification of genetic anomalies such as tnsoπues and translocations

The dyes of the mvention are generally not quenched by add g a halogenated deoxyundine (preferably bromo- or chloro-deoxyundme) Therefore, dyes that overlap spectrally with bisbenzimidazole dyes (e g Hoechst 33258, Hoechst 33342, Molecular Probes, Oregon) and bmd to nucleic acids m close proximity to the bisbenzimidazole dyes can be used m conjunction with halogenated deoxyundines and bisbenzimidazole dyes for the analysis of cell proliferation The effect of the additional reagents m combmation with the dyes of the mvention is analysed by fluorescence quantifying instrumentation (Example 49)

Additional dyes are optionally used to differentiate cells or cell-free samples containmg nucleic acids according to size, shape, metabolic state, physiological condition, genotype, or other biological parameters or combmations thereof The additional reagent is optionally selective for a particular charactenstic of the sample for use m conjunction with a non-selective reagent for the same charactenstic, or is selective for one charactenstic of the sample for use m conjunction with a reagent that is selective for another charactenstic of the sample In one aspect of the mvention, the additional dye or dyes are metabolized lntracellularly to give a fluorescent product mside certain cells but not inside other cells, so that the fluorescence response of the cyamne dye of the mvention predominates only where such metabolic process is not tak g place Alternatively, the additional dye or dyes are specific for some external component of the cell such as cell surface proteins or receptors, e g fluorescent lectins or antibodies (Example 32) In yet another aspect of the mvention, the additional dye or dyes actively or passively cross the cell membrane and are used to mdicate the mtegnty or functioning of the cell membrane (e g calcein AM or BCECF AM) In another aspect, the additional reagents bmd selectively to AT-nch nucleic acids and are used to mdicate chromosome banding In another aspect of the mvention, the additional reagent is an organelle stam, l e a stam that is selective for a particular organelle, for example the additional reagent(s) may be selected for potential sensitive uptake mto the mitochondπa (e g rhodamine 123 or tetramethylrosamine) or for uptake due to pH gradient m an organelle of a live cell (e g Diwu, et al , CYTOMETRY supp 7, p 77, Abstract 426B (1994))

The additional dyes are added to the sample m an effective amount, with the optimal concentration of dye determmed by standard procedures generally known the art Each dye is optionally prepared m a separate solution or combmed m one solution, depending on the intended use After illummation of the dyed cells at a suitable wavelength, as above, the cells are analyzed according to their fluorescence response to the illummation In addition, the differential fluorescence response can be used as a basis for sorting the cells or nucleic acids for further analysis or experimentation For example, all cells that "survive" a certain procedure are sorted, or all cells of a certain type m a sample are sorted The cells can be sorted manually or usmg an automated technique such as flow cytometry, according to the procedures known m the art, such as m U S Patent 4,665,024 to Mansour, et al (1987)

The examples below are given so as to illustrate the practice of this mvention They are not mtended to limit or define the entire scope of this mvention

Example 1 The following compound is prepared

The synthetic precursor (1) is prepared accordmg to Example 1 of U S Patent No 5,436,134 to Haugland et al (1995)

Example 2 Preparation of 2-chloro-4-methyl-l -phenylquιnolιnιum chlonde (2) The following compound is prepared

To 2 8 g (1 1 9 mmoles) of 1 m 20 mL of methylene chlonde is added 1 85 g of POCl ₃ and a catalytic amount of dimethylformamide (Marson, TETRAHEDRON , 48, 3659 (1992)) The resulting mixture is heated to reflux for 24 hours The crude product is used without further purification, or is purified usmg column chromatography

The methoxyqumolimum analog is prepared m the same way, except usmg 1 ,2-dιhydro-7-methoxy-4-methyl- 1 -phenyl-

2-quιnolone m place of 1

Example 3 Preparation of Dve 640

The commercially available 2-chloro-3-methylquιnolιne is methylated by heating with an excess of methyl iodide m a sealed tube at 120 °C for one hour At the end of the reaction, ethyl acetate is added and the precipitate is filtered to isolate the qumolmium iodide This intermediate compound is then stirred with 3-methyl-2- methylthiobenzothiazohnium tosylate m methylene chlonde in the presence of one equivalent of tπethylamme to yield the desired product

Example 4 Preparation of 2-chloro-4- 2.3-dιhvdro-3-methyl-.benzo-1.3-thιazol-2-ylVmethvhdenel-l - phenylquinolinium iodide ("31

The following compound is prepared

Compound 3 is prepared accordmg to Example 7 of U S Patent 5,436,134 to Haugland et al (1995)

The pyndinium analog of Compound 3 is prepared m the same way, except usmg the pyndimum analog of 2

The tnmethme dye analog is prepared similarly, except usmg 2-(2-aml ovmyl)-3-methylbenzothιazohum tosylate m place of 3-methyl-2-methylthιobenzothιazohum tosylate

Example 5 Preparation of Dve 937 Dye 937 is prepared by heating 3 at 55 ^C C m the presence of N-(3-dιmethylammoproρyl)-N-propylamme m 1 ,2- dichloroetheane for two hours

A family of analogous ammoalkylammo-substituted dyes are prepared similarly, by treating the appropπate 2-chloro denvative with a selected amine (For example Dyes 21 1 , 298, 342, 377, 396, 397, 856, 938, 993, 1004, 1 168 and 10101)

Example 6 Preparation of Dve 1 107

Dye 937 is treated with an excess of methyl iodide and PROTON-SPONGE (Aldnch) to methylate the dimethylamme and give the quaternary ammomum salt A family of analogous ammomumalkylammo-substituted dyes are prepared smularly, by treated an appropπate ammoalkylammo-substituted dye (See Example 5) with methyl iodide and

PROTON-SPONGE (For example, Dyes 308, 309, 345, 398, 1107, 1114, 1 170, 1 172, and 3102)

Example 7 Preparation of Dve 1004

2-Dιmethylaιιunoethanethιol is added to 2-c__loro-4-[2,3-dihydro-3-methyl-(benzo-l,3-oxazol-2-yl)-me thylidene]-l- phenylquinolinium iodide (the benzoxazohum analog of 3) m methylene chlonde, followed by tnethylamine, and the resulting mixture is stirred at room temperature for 1 5 hours The volume of solvent is reduced under reduced pressure and the product is isolated by filtration

A family of analogous aπunoalkylthioether-substituted dyes is prepared smularly, by treatmg the appropnate 2 -chloro denvative with a selected aminoalkylthiol in the presence of one equivalent of tnethylamine (For example Dyes 365,

380, 387, 996, 1004, and 1 169) The resulting dyes are quatemized usmg the method of Example 6 to yield the correspondmg ammomumalkylthioether-substituted dyes (For example Dyes 352, 391 , 1155, and 1167)

Example 8 Preparation of Dves 314 and 316 The l,2-dihydro-4-methyl-l-phenyl-2-qumolone is heated at reflux with 1 3 equivalents of phosphorus oxychlonde and

1 equivalent of DMF in toluene for one hour to generate the 2-chloro-4-methyl-l -phenylquino nium chlonde (3) The chlonde is then stirred m the correspondmg dimethylaminoethanethiol m methylene chlonde to produce the correspondmg 2-dιmethylanunoethylthιo- 1 -phenylquinolinium chlonde This is then reacted with one equivalent each of the 2-(2-amlmovmyl)-3-methyl-benzoxazohum tosylate, tnethylamine and acetic anhydnde to generate the correspondmg tπmethme denvative

The dimethylamino denvative is quatemized usmg excess methyl iodide and PROTON-SPONGE to yield Dye 316

Example 9 Preparation of Dve 381 The starting 1 ,2-dιhydro-4-methyl- 1 -(4'-methoxyphenyl)-2-quιnolone is prepared by an Ullmann reaction of the 2- hydroxy-4-methylquιnolιne with 4-ιodoanιsole The methyl ether is demethylated with boron tπbrormde and the resulting phenol is alkylated m acetone with 3-dιmethylanunopropyl chlonde and potassium carbonate to yield the dimethylammoalkylether qumolone To this qumolone in THF at -78 °C is added 3 equivalents of n-butylhduum After one hour at low temperature the reaction is quenched with 5 equivalents of acetic acid and allowed to warm to room temperature, where it is stirred for an additional several hours The volatile components are removed under vacuum and the resulting crude qumolmium salt is stirred with 3-methyl-2-methylthιobenzothιazohum tosylate m methylene chlonde m the presence of tnethylamine to generate the correspondmg 2-butyl-l-((3'-dιmethylammopropoxy)phenyl)-cyanme, which is quarteπuzed as above with methyl iodide and PROTON-SPONGE to yield the desired product

Example 10 Preparation of Dve 374

The procedure is similar to that used to prepared Dye 381 (Example 8) except that l,2-d-hydro-7-(3'- d_methylammopropoxy)-4-methyl-l -phenyl-2-qumolone is used as the starting matenal instead of l,2-dιhydro-4-methyl- l-(4'-(3"-dιmethylammopropoxyphenyl))-2-q_unolone

Example 1 1 Preparation of Dve 3100

The following compound is prepared

2,4-Luudιne is heated with methyl iodide in a sealed tube at 100 °C to generate the pyndimum iodide, which is then treated with the 3-methyl-2-methylthιobenzothιazolιum tosylate m the presence of one equivalent of tnethylamine to generate the desired product

Example 12 Preparation of Dve 3103

The correspondmg 2-chloro denvative is heated at about 90 °C, m a sealed tube, a 1 1 v/v mixture of chloroform/methanol for 10 hours to yield the desired product

Example 13 Preparation of Dve 388 To 1 ,2-dιmethyl-4-qumolone m THF at -78° C, is added 3 equivalents of 4-dιethylaιιunomethylphenylhthιum The reaction mixture is stirred at room temperature for one hour, after which 5 equivalents of acetic acid is added, the mixture is warmed to room temperature and stirred for an additional 3 hours All the volatile matenals are removed under vacuum and the crude residue is stirred with one equivalent each of 3-methyl-2-methylthιobenzothιazolιum tosylate and tπethylamme m methylene chlonde to yield the diethylanunoalkyl denvative This is quartemized directly with excess methyl iodide and PROTON-SPONGE to yield the desired product

Example 14 Preparation of Dve 390

Dye 390 is prepared analogously to Dye 388, except usmg 1 ,2-dιhydro- 1 ,4-dιmethyl-2-quιnolone m place of 1 ,2- dunethyl-4-qumolone

Example 15 Preparation of Dve 380

4-Dιmethylamιnobutyryl chlonde is treated with one equivalent of 5-ammo-l,3,4-thιadιazole-2-thιol (Aldnch) m the presence of tnethylamine to generate the correspondmg amide thiol This intermediate product is then treated with 2- chloro-4-[2,3-dιhydro-3-methyl-(benzo- 1 ,3-thιazol-2-yl)-methγhdene]- 1 -phenylqumolium iodide to yield the desired product

Example 16 Preparation of Dve 1 189

A solution of 2-chloro-4-(2,3-dιhydro-3-methyl-(benzo-l,3-thιazol-2-yl)- methylιdene)-l-phenylqumohum iodide is treated with 4-aπunothιophenol to yield the correspondmg 2-(4'-a__ι_nothιophenoxy) denvative The aniline is then reacted with 4-bromobutyτyl chlonde (Lancaster) to yield the 4-bromobutyramιde This intermediate is heated with excess pyndine to yield the final product

Example 17 Preparation of Dve 517

To l,2-dιmethyl-4-methoxy-quιnolιnιum iodide m methylene chlonde is added one equivalent each of 2-(2- amlmovmyl)-3-methylbenzothιazohum tosylate, tnethylamine and acetic anhydnde, m that order The reaction is stirred at room temperature overnight to yield the product

Example 18 Preparation of Dve 300 The following compound is prepared

2,4-dιmethylqumolme is heated with 10 equivalents of 1,3-dnodopropane, neat, at 150 ^C C to generate the qumolmium iodide The iodide is then reacted with 3-methyl-2-methylthιobenzoxazohum tosylate m the presence of one equivalent of tnethylamine to generate the 1 -lodopropyl intermediate, which m turn is transformed to the final product by heating with a large excess of tnmethylamme m a sealed tube at 100 °C

Example 19 ds DNA m electrophoretic gel

A dilution senes of φX174 rep cative form (double-stranded) bacteπophage DNA digested with either Hae III restπction endonuclease or λ cI857 bacteπophage DNA digested with Hind III restπction endonuclease (both DNAs available commercially) is prepared in 10 mM Tns-HCl, pH 7 5, 1 mM ethylenediamine tetraacetic acid (EDTA) (TE) An equal volume of 15% FICOLL is added to each sample and samples are loaded onto a 5% polyacrylamide gel for the Hae III digest or to a 1 % agarose gel for the Hind III digest Electrophoresis is earned out under standard conditions The resultmg gels are transferred to small staimng dishes containmg a 1 μM solution of Dye 377 m 89 mM Tπs base, 89 mM bone acid, 1 mM EDTA, pH 8 (TBE) The stammg solution is then covered with foil to protect it from room light and agitated gently for 15-30 mmutes The gels are then transferred directly to a transillummator and photographed usmg 300 nm transillumination or 254 nm epi-illumination, black and white Polaroid 667 pnnt film and a Wratten 15 gelatin filter DNA appears visually as bnght green fluorescent bands

Example 20 ss nucleic acids in electrophoretic gels

Native or denatured electrophoretic gels of a dilution senes of £ coli nbosomal RNA, Ml 3 single-stranded DNA or a synthetic oligonucleotide are prepared using standard methods Gels are then stained with 1 μM Dye 388 m TBE and visualized as m Example 19 RNA and DNA bands appear as bnght green fluorescent bands

Example 21 Ouantitation of double-stranded DNA in solution

A senes of double-stranded DNA samples of unknown concentration is prepared m TE A working solution of 0 8 μM Dye 377 is prepared in TE and kept protected from light One mL of each DNA solution is placed m a fluorescence cuvette One mL of working dye solution is added to each cuvette, the samples are mixed and mcubated 2 to 5 mmutes, protected from light Fluorescence is measured in a standard fluorometer or microtiter plate reader, usmg 485 nm excitation light and measuπng the emission at 520 nm Fluorescence intensity is compared to a standard curve prepared from samples containmg known DNA concentrations The concentration of DNA the unknown samples is determmed by mteφolation of the data in the standard curve Samples containmg DNA m excess of 1 μg/mL are diluted pnor to quantitation The assay is lmear between about 25 pg mL and 400 ng/mL in DNA concentration, as shown m Figure 3 The assay is about 20-fold more sensitive than can be achieved with either YOYO-1 or YO-PRO-1 (0 5-1

ng/mL), about 400-fold more sensitive than Hoechst 33258 (10 ng/mL) and about 40,000-fold more sensitive than UV absorbance measurements (~1 μg/mL)

Example 22 Ouantitation of single-stranded o gonucleotides in solution A senes of single-stranded synthetic ohgonucleotides, synthesized from standard or moφholine-modified denvatives

(AntiVirals Inc , Corvalhs, OR), at least 8 bases m length, m solutions of unknown concentration are diluted to 1 mL m TE in fluorescence cuvettes One mL of a 0 5 μM solution of Dye 309 in TE is added to each sample, and the samples are mcubated for 2-5 mmutes at room temperature, protected from light The samples are illuminated at 485 nm and the fluorescence of each sample is measured at 520 nm The concentration of each solution is determmed by companson with a standard curve prepared using known amounts of ohgonucleotides, as shown in Figure 4 Samples containmg m excess of about 1 μg/mL nucleic acid are diluted pnor to analysis Samples containmg as little as about 100 pg/mL synthetic oligonucleotide with standard bases and links can be assayed Samples contammg moφholine modified links are detected at lower sensitivity, with such sensitivity bemg a function of sequence Ohgonucleotides of at least 8 bases m length can be measured This sensitivity is greater than 10,000 times more sensitive than measurement of UV absorbance, which is the current method most commonly used for oligonucleotide detection and quantitation

Example 23 Detection of ohgonucleotides m blood

Whole blood is collected in vials contammg EDTA 0 5 mL aliquots of blood are transferred to 1 5 mL rmcroftige tubes

To each sample is added a 24 base oligonucleotide, m as small a volume as possible ( 1 to about 50 μL), m amounts ranging from 1 ng total up to about 10 μg total Blood cells are pelleted by centrifugation m the microfuge for 1-2 mmutes at 5000 φm, at room temperature The supernatant liquid is removed to fresh tubes, without disturbing the pellet. Remaining cells are removed by recentrifugation for 1-2 mmutes at 10,000 φm, at room temperature The supernatant liquid is again carefully transferred to fresh tubes, without disturbing the pellet An equal volume of phenol CHC1 ₃ isoamyl alcohol, 24 24 1 is added to each tube, and the tubes are vortexed vigorously and centπfuged m the microfuge to separate phases (room temperature) The aqueous layer is removed to fresh tubes, carefully avoiding the interface The extraction is repeated Aliquots contammg 200 μL of each sample are transferred to fluorescence cuvettes containmg 800 μL TE One mL of a 0 5 μM solution of Dye 309 m TE is added to each cuvette and samples mixed by inversion The amounts of ohgonucleotides present are determmed by subtracting the fluorescence observed from a control sample contammg no oligonucleotide, accordmg to the method outlmed m Example 22

Example 24 Detection of DNase activity

Samples thought to exhibit DNase activity are mcubated at 37 °C for five mmutes with 10 ng of φX174 RF (double- stranded) DNA, digested with Pst I restπction endonuclease, a buffer consisting of 50 mM Tπs-HCl, pH 7 5, 10 mM MgCl ₂ , 1 mM CaCl ₂ , and 50 μg/mL bovine serum albumin m a total volume of 10 μL Reactions are quenched by the addition of 2 5 μL 100 mM EDTA and immediate vigorous mixing An equal volume of 15% FICOLL is added to each sample and the samples are mixed bneflv, then loaded onto a 1% agarose minigel along with molecular weight markers containmg 0 05% bromophenol blue tracking dye and 7 5% FICOLL The gel is electrophoresed under standard conditions, until the bromophenol blue has migrated at least 1 1/2 to 2 mches The gel is then removed from the electrophoresis apparatus and placed in a stainmg dish A solution containmg 1 μM Dye 377 m TBE is added to the gel

and the gel is agitated gently, protected from light, for at least 20 mmutes The gel is transferred to a transillummator, illuminated with 300 nm transillummation or 254 nm epi-illumination and photographed with Polaroid 667 black and white pnnt film, through a Wratten 15 gelatm filter DNase activity appears as smearing of the smgle, shaφ Pst I digested DNA band As little as 6 5 pg or ~2 x 10 ^'5 units of DNase I can be detected m this way Restiiction endonuclease activity or RNase activity can be assayed m a similar manner, usmg appropπate substrate nucleic acid molecules

The assay is generalizable for topoisomerases, gyrases, restπction endonucleases, RNases, exonucleases, or any enzyme that acts on DNA m such a way that its electrophoretic mobility is altered

Example 25 Detection of nucleic acids on a support

Plasmid pUC 19 DNA is digested oveπught with a smgle restiiction endonuclease or a mixture of two enzymes One μg of each sample is then loaded onto a 5% polyacrylamide gel and electrophoresed accordmg to standard procedures The gel is then stained with Dye 398 as descnbed m Example 19 above Bands are visualized by U V illumination, and the nucleic acids m the bands are denatured and electrophoretically transferred to a nylon membrane After transfer, green fluorescent DNA bands are visualized usmg a hand-held UV lamp, due to the retention of Dye 398 The membrane is prehybπdized, hybndized and washed accordmg to standard procedures, usmg a biotin-labeled Ml 3 sequencing primer (specific for the lacL gene) Hybndized bands are detected usmg streptavidin alkaline phosphatase along with NBT/BCIP substrate All fragments that contam a primer bmdmg site show specific hybndization signals (a bluish puφle color) In addition, the presence of the dye also does not affect on the efficiency of hybndization, since an identical (control) gel that is not stained but is blotted and hybndized at the same time exhibits identical signals The dye signal is lost during hybndization, so the blot is restained to visualize all of the DNA bands RNA can also be detected on filter membranes by stammg with an appropπate dye

Example 26 Counterstaming metaphase chromosomes and inteφhase nuclei

Human metaphase chromosome spreads are prepared accordmg to standard procedures Spreads are denatured, prehybπdized and hybndized, accordmg to standard procedures, to alpha centromere repeat probes that have been labeled with a biotin-labeled nucleotide tπphosphate by random priming The hybndized probes are then detected by further labeling with TEXAS RED fluorophore-labeled streptavidin (Molecular Probes, Inc , Eugene OR) and are counterstained by applymg a 1 μM solution of Dye 1 1 14 m phosphate-buffered salme (PBS) Samples are mounted, coverslips sealed and stained chromosomes are visualized with a fluorescence microscope and a fluorescein filter set to see the counterstam and a filter set appropnate for the TEXAS RED fluorophore to visualize the centromere signal This assay can be generalized to be used with a fluorophore label, either on the nucleoside tπphosphate or the streptavidm, that is spectrally distinct from the counterstam

Example 27 Chromosome banding

Human metaphase chromosome spreads are prepared accordmg to standard procedures The covershp is πnsed with PBS, then stained with 0 1 μM Dye 1 1 14 in 0 1 M sodium phosphate, pH 6 5 for 30 mmutes at 37 °C and πnsed with PBS Chromosomes are then counterstained with 10 mg/mL Methyl Green the same buffer, for 30 mmutes at 37 °C

The slide is rinsed twice m PBS and then mounted m 10% PBS contammg 1 mg mL p -phenylenedianune and 78% glycerol Chromosomes contammg bands are observed through a fluorescence microscope equipped with a standard fluorescein filter set

Example 28 Detection of protein DNA complexes in gels using pre-labeled DNA templates

Single-stranded Ml 3 phage DNA is mcubated, in an appropπate bmdmg buffer, with protems required for T4 phage replication as follows g41 p (hehcase), g61 p (pnmase) and g41 p m the presence of g61 p Samples are mcubated for sufficient time for complex formation then electrophoresed on an agarose gel usmg a ninnmg buffer that is optimized for DNA protem complex formation The gel is stamed with Dye 377 as descnbed m Example 19 above DNA containmg bands are visualized directly usmg 254 nm epi-illumination or 300 nm transillummation Samples contammg pnmase alone or pnmase plus hehcase result in shifted electrophoretic mobility complexes m companson with samples containmg no protein at all The hehcase does not yield a shifted complex by itself This assay can be generalized to detect bmdmg of any nucleic acid bmdmg protein or factor that causes a shift in the electrophoretic mobility of the template upon bmdmg

Example 29 Detection of sequence-specific DNA binding proteins in cell extracts

DNA templates of about 25 to about 200 base pairs m length, contammg sequences of interest are mcubated with Dye 1 1 14 at a dye bp ratio of 1 30, m the dark, at room temperature A DNA template that is virtually identical, except for lackmg the test sequence is labeled and treated in parallel Extracts are prepared from cells of interest usmg standard techniques Approximately 1 ng to 1 microgram of DNA is mcubated with about 15 πucrograms of protein from the crude extract, in the presence of about 2 micrograms of poly(dI-dC)-poly(dI-dC) earner nucleic acid and bovine serum albumin m a buffered solution The sample is mcubated for about 15 minutes at about 30 °C, m the dark Generally a titration of extract must be tested m order to determme the optimal concentration for detection of specific bmdmg interactions FICOLL or glycerol is added, to a final concentration of about 5-7 5% and the samples loaded onto a polyacrylamide gel that is cast usmg low ionic strength buffers A sample containmg bromophenol blue tracking dye and FICOLL or glycerol alone is loaded m parallel Samples are electrophoresed until the bromophenol blue has run at least a few inches mto the gel The gel apparatus is disassembled and the green fluorescent bands directly observed following illumination usmg 254 nm or 300 nm U V light, or a laser scanner with —490 nm excitation light and ~530 nm collection filters Extracts containmg sequence-specific b dmg factors that recognize the template of interest will yield bands of shifted mobility with respect to other extracts and the combmation of such extracts with the control DNA template

Example 30 Preparation and use of prelabeled marker DNA

The same labeling technique of Example 19 is used to stain unlabeled DNA markers to prepare prelabeled DNA markers Some bands are visible in the presence of ordinary fluorescent room light alone or visualized as above The position of the bands mdicates the distance that the samples have migrated and can be used to determme the size of other DNA molecules that are electrophoresed in tandem on the same gel

Example 31 Detection of nbosomal RNA in sucrose gradients

Mammalian cells are grown under standard conditions RNA is prepared usmg standard protocols A gradient of sucrose ( 10-40% w/v) is prepared m a buffer contammg 20 mM Tns-Cl, pH 80, 5 mM EDTA, and 1 μM Dye 388 m polypropylene centrifuge tubes In a volume of 0 5 mL or less, the RNA is carefully layered on top of the gradient Gradients are kept protected from light The tubes are loaded mto a Beckman SW28 (or equivalent) rotor and centnfiiged at 26,000 φm for 24 hours at 15 °C The tubes are carefully removed from the rotor and the nucleic acids visualized as bπghtly green fluorescent bands usmg a handheld UV lamp Ribosomal RNA's are visible as three mdependently migrating species, the most rapid is the 23S, the next is the 16S and the slowest are the 5S species, and tRNA's Nucleic acids are collected by piercmg the bottom of the gradient, usmg a 21 gauge needle Small (0 5 mL) fractions are collected and aliquots of each analyzed by gel electrophoresis m companson with RNA's of known size

Example 32 Counterstainmg fixed tissue culture cells that have been probed an additional detection reagent Mouse fibroblast cells (NIH 3T3) are grown under standard conditions Cell media is removed and the cells washed bnefly with HBSS (Hanks balanced salt solution with magnesium and calcium) Cells are fixed with 3 7% formaldehyde m HBSS and washed three times more with PBS Cells are permeabilized with 0 1% TRITON X-100 m PBS, with agitation for 5 mmutes Cells are nnsed three times with PBS, blocked by incubation with 2% fetal calf serum, 0 1 % Tween 20 m PBS for 30 minutes to an hour A rabbit antibody directed against Golgi membranes is applied m blocking solution, for one hour Cells are πnsed m PBS and then mcubated with an anti-rabbit secondary antibody that has been conjugated to TEXAS RED dye (diluted m blocking buffer), then washed again m PBS To counterstam, a solution containmg 0 4-0 01 μM Dye 1 1 14 is applied to the cells and they are mcubated for 10 mmutes at room temperature Cells are washed bnefly m PBS and visualized with a fluorescence microscope and standard fluorescem filters to visualize counterstainmg and through a filter set for TEXAS RED dye to visualize Golgi stainmg Nuclei show bnght green fluorescence and the cytoplasm appears slightly dimmer green

Example 33 Detection and quantitation of DNA amplification products Target-specific primers contam hapten or fluorophore labels on their 5' ends that are biotin, dinitrophenyl, a fluorescem fluorophore, a BODIPY FL fluorophore, a BODIPY TMR fluorophore, or a BODIPY TR fluorophore (Molecular Probes, Eugene OR) Target DN A-contaimng samples are combmed with primer pairs m the presence of appropnate buffers and samples are amplified according to optimal conditions for each pπmer pair Such conditions must be determmed empincally D A amplification products are then detected by either loading aliquots of samples onto agarose or polyacrylamide gels followed by stammg as m Example 19, or by a solution assay as m Example 21 The amount of the DNA amplification product present at the end of the amplification reaction is a direct mdicator of the amount of target present in the ongmal sample, thus it can be used to assay target number, even when such numbers are too low to assay by direct application of the technique descnbed m Example 21 The enhanced sensitivity of the present dyes also allows analysis of amplification products after fewer amplification cycles This procedure is illustrated schematically m Figure 2

Example 34 Detection of single-strand conformation polvmoφhisms

DNA amplification products with sizes ranging from 100-250 base pairs, containmg sections of human p53 and K-ras genes are prepared from human gastnc adenocarcinomas as descnbed by Perkins, et al (NUCLEIC ACIDS

RESEARCH, 21, 3637 (1993)) About 20-100 ng of the DNA amplification products, m a volume of 5 μL, is mixed with 0 4 μL of 1 M methylmercury hydroxide, 1 μL of 15% FICOLL and 13 6 μL of TBE buffer The mixture is heated to 85 °C for 4 mmutes and then quickly chilled on ice A 20% polyacrylamide gel prepared usmg TBE buffer, pre- equi brated at a set temperature (which must be determined empmcally for each sample) and the samples loaded onto the gel along with a sample contammg only FICOLL and tracking dye The gel is electrophoresed under constant temperature control until the marker dye is close to the bottom of the gel The gel is stamed with a 1 μM solution of Dye 377 m TBE as descnbed m Example 19 and visualized usmg 254 nm-300 nm UV illummation DNA molecules that differ m sequence appear as bands with distmct separate mobilities The presence of bands with different mobilities is therefore indicative of even smgle pomt mutations m the target genes

Example 35 Determination of superhe cal state by gel electrophoresis

Closed circular DNA is prepared usmg standard procedures Samples of closed circular DNA and size marker DNA samples are applied to a senes of 0 7% agarose minigels containmg dyes m the concentration range of 001 μM to about 1 μM Samples are electrophoresed until the circular forms have migrated at least half of the length of the gels Gels are then visualized directly usmg ultraviolet illummation or are poststamed with Dye 377 and then visualized, as descnbed in Example 19 Closed circular samples generally contain both supercoiled and relaxed DNA molecules Treatment with enzymes such as topoisomerases or gyrases can change the topological characteπstics of closed circular DNA, such as the number of supercoils present m a given molecule If intercalating dyes such as ethidium bromide are bound to templates with negative supercoils versus relaxed DNA molecules, more dye molecules bmd to the negatively supercoiled template than to the relaxed molecule In addition, as negatively supercoiled DNA is titrated with ethidium bromide, the molecule passes from a negatively supercoiled form to relaxed DNA and then finally becomes positively supercoiled These three different topological forms are characterized by their migration m electrophoretic gels In general, supercoiled DNA's migrate more rapidly than identically sized relaxed molecules or lmear DNA's There is a cntical concentration of ethidium bromide that induces the change from negatively supercoiled to relaxed to positively supercoiled DNA This concentration for ethidium bromide occurs at about 0 1 to 0 5 μg mL dye The dyes of the present mvention, such as Dye 377 can also cause this change m topological form Since these dyes allow detection of much less DNA m a band on a gel, they provide a much more sensitive assay than do dyes such as ethidium bromide for this application In addition, the new dyes can be used m combmation with ethidium bromide, as sensitive poststains Thus Dye 377 can be used to probe the topological state of closed circular DNA molecules and can therefore be used to assay topoisomerase or gyrase activity on such templates

Example 36 Labels for microiniection of DNAs

Plasmid DNA is labeled with Dye 11 14 by incubation for at least five mmutes at room temperature, protected from light, with a solution contammg no more than 1 dye molecule per 5 base pairs of DNA DNA is microinjected mto cells usmg standard techniques (Noueiry et al , CELL, 76, 925 (1994)) Labeled DNA appears as bnght green fluorescence in cells, usmg a fluorescence microscope fitted with a fluorescem filter set

Example 37 Labeling and detection of single DNA molecules

Individual phage lambda DNA molecules are tethered to microscope slides by either tethering one end through biotin/streptavidm linkages or polylysine spread bmdmg (Perkins et al SCIENCE 264, 822 (1994), Perkins et al , SCIENCE, 264, 819 ( 1994)) A solution contammg 10 μM Dye 1 1 14 m TE, with 2% β-mercaptoethanol is applied to the slide Covershps are mounted the presence of the dye staimng solution and a mounting medium Smgle stamed DNA molecules can be observed m the fluorescence microscope with a standard fluorescem optical filter set Molecules can be spread or stretched usmg optical tweezers (Perkins et al and Perkins et al supra, Bensimon et al., SCIENCE 265, 2096 (1994)) Smgle nucleic acid molecules can also be detected and sized m a flow cytometer following stainmg with this dye, m a manner analogous to that used for stainmg with TOTO-1 nucleic acid stam (Goodwin et al , NUCLEIC ACIDS RESEARCH 21, 803 (1993), Castro et al , ANAL CHEM 65, 849 (1949))

Example 38 Ouantitation of cell number

Tissue culture cells are grown under standard conditions Cells are harvested by centπfiigation for nonadherent cells and by trypsinization followed by centrifugation and washing in PBS for adherent cells Cell pellets are lysed by suspension in 100 μL of a solution of 0 1 % TRITON X- 100 detergent m water Cell lysates are diluted to 1 mL with TBE and then added du-ectly to 1 mL of a 0 8 μM solution of Dye 410 m TBE and mixed Samples are mcubated about 5 mmutes m the dark and then fluorescence at 520 nm is measured following excitation at 485 nm usmg a standard fluorometer The mtensity of the fluorescence emission is directly proportional to the amount of double-stranded DNA present, which is directly proportional to the cell number, as shown Table 10 below Fluorescence emission is compared directly with a standard curve made from known amounts of DNA (as descnbed m Example 21, Figure 3) to determme the amount of DNA present and is compared with results from a standard curve prepared with known quantities of the identical type of cell m order to directly assay for cell number While the dynamic range of this assay is exceptionally large, as shown m Figure 5 A, as few as 5-10 cells/mL can be detected usmg this procedure, as shown m Figure 5B) The dyes can also be used m this way to assay reagents, drugs or hormones that either inhibit or enhance cell proliferation

Table 10 Relationship between cell number and DNA content

Example 39 Discπmination of RNA. ds DNA and ss DNA using nucleases in combmation with fluorescent dves Samples contammg either RNA, double-stranded DNA or single-stranded or combmations of these nucleic acids m concentrations of about 100 pg/ mL to about 1 μg mL are mcubated mdependently with the following reagents a) DNase I (which digests double-stranded DNA), b) RNase A and Tl Nuclease (which digest RNA), c) mung bean nuclease (which digests single-stranded DNA) or d) RNase H (which digests DNA/RNA hybπds and some double- stranded RNA's) m the presence of the appropπate buffer for each enzyme In addition, control samples that are not subjected to enzymatic digestion are prepared After digestion is allowed to go to completion, samples are added to cuvettes contammg 0 4-0 8 μM of a dye of the present mvention, such as Dye 309, samples are then mixed and mcubated 5 mmutes m the dark Fluorescence mtensity is measured m a fluorometer The type of nucleic acid present m the sample is determmed usmg Table 1 1 If a sample yields fluorescence (mdicated by + m the table) equal to the amount yielded by the undigested control, then it does not pπmanly consist of the nucleic acid targeted by the enzyme This set of data can be used to determme the amount of each species of nucleic acid present m a mixed sample, usmg standard curves generated with pure double-stranded DΝA, single-stranded DΝA, RΝA and RΝA DΝA hybπds

Example 40 Discπmmatioπ of ds DNA from ss DNA using fluorescent dves

Two nucleic acid samples are prepared havmg concentrations of less than 0 2 μM The first sample is mixed with a monomethine dye of the present mvention to a final concentration of 0 2 μM dye ( 1 1 ratio) in TE in a fluorescence cuvette The second sample is mixed with the same dye to a final concentration of about 1 μM or higher, m TE buffer a fluorescence cuvette Both samples are mcubated for at least 5 mmutes at room temperature m the dark A fluorescence emission spectrum is generated for each sample, following excitation at about 485 nm, usmg a standard fluorometer Samples containmg only double-stranded DNA yield fluorescence emission spectra with maxima in the green wavelengths, at about 500-535 nm at both dye base ratios Samples contammg only single-stranded DNA, however, yield a fluorescence spectrum with a maximum m the green (at about 500-535 nm) only when the dye base ratio is less than 1 1 At dye base ratios greater than 1 1 the emission maxima for smgle stranded nucleic acids shifts to longer wavelengths (typically 550-580 nm)

Some of the dyes, such as Dye 377, have very low intensities for the longer wavelength emission and appear to simply lose the green fluorescence Nucleic acids at a final concentration of 1 5 nM bases are mcubated with Dye 377 at a concentration of 0 8 μM Calf thvmus DNA was used as the double-stranded molecule (ds DNA) and Ml 3 phage DNA

was used for smgle-stranded DNA (ss DNA) The maximum emission wavelength for double-stranded DNA is at ~520 nm, but for smgle-stranded DNA under these conditions the peak emission is at ~550 nm (as shown m Figure 6)

Others, such as Dye 1 1 14, have significant longer wavelength signals that are almost as intense as the green emission , and double-stranded and smgle-stranded nucleic acids can be discriminated m cells Ethanol-killed E coli cells are suspended m water at a concentration of ~10 ⁸ cells/mL Three bacterial suspensions are then mcubated at room temperature with Dye 1 1 14 at concentrations of 0 1 μM, 0 5 μM and 1 0 μM respectively Followmg stainmg, the samples are illuminated at 480 nm and the fluorescence emission recorded from 490 nm to 700 nm, as shown m Figure 7 At low sta mg concentrations (0 1 μM) the fluorescence response is pnmaπly a strong green fluorescence (-520 nm) As the staimng concentration increases (0 5 μM), the green fluorescence mtensity increases somewhat, with an accompanying mcrease in red fluorescence (-630 nm) As dye concentration continues to mcrease ( 1 0 μM), the red fluorescence mtensity matches the now-decreasmg green fluorescence The red fluorescence emission is due to the presence of smgle-stranded nucleic acids present m the stamed E coli

Example 41 Base selectivity of selected dves

Synthetic homopolymers of πbo- or deoxynbo- nucleic acids are mcubated at concentrations of 20-50 μM with Dyes 937, 1004, 993, 309, 396, 410, respectively, at concentrations of about 1 μM m TE, for about 5 mmutes, at room temperature, in the dark Fluorescence emission at about 500-530 nm is measured m a fluorometer for each sample followmg excitation at 485 n Certam of the dyes show pronounced selectivity m fluorescence accordmg to the nature of the homopolymer as shown m Table 12 Thus these dyes can be used m combmation with other dyes, such as

Hoechst 33258 (which is AT selective) to determme information about primary nucleic acid structure

Table 12 Base selectivity of selected dyes

Quantum yields are shown for several dyes bound to several different nucleic acid substrates Poly πbo G probably shows extremely high quantum yields because it has formed higher order structures such as tπple-stranded molecules, rather than as a result of base selectivity Poly dl is a polymer of inosine, which behaves much like guanine m nucleic acids

Example 42 Detection of viable bactena in a food sample

One gram samples of ground beef are agitated with 9 mL of stenle water at medium speed m a vortexer for 1 minute Three 0 1 mL aliquots are removed and spread uniformly over the surface of three 100 mm eosin-methylene blue plates, which are subsequently mcubated for 24-48 hours at 37 °C An 800 μL aliquot is removed and 200 μL of 5% bovine serum albumin m stenle distilled water are added 1 μL of a 5 mM DMSO solution of Dye 345 and 100 μL of a 1 mg/mL solution of rabbit antι-0157 H7 IgG are added to the sample, which is then mcubated for 15 mmutes at room temperature with slow mixing The sample is then washed by centn_ugatιon at 10,000 x g for 20 sec m a 1 5 mL tube, and resuspended m 1 mL of stenle water with 4% glutaraldehyde After 15 mmutes mcubation at room temperature, the bactena are pelleted by centrifugation as above and resuspended m 1 mL of stenle water 2 μL of a 1 mM DAPI solution m DMSO, 1 μL of 5 mM Dye 345, and 20 μL 1 mg/mL TEXAS RED fluorophore-conjugated goat anti-rabbit

IgG are subsequently added and the sample is mcubated for 15 mm at room temperature with slow πuxmg Live bactena are blue fluorescent and dead bactena are green fluorescent Only enteropathogenic E coli are red fluorescent

Example 43 MIC determination of an antibiotic using flow cvtometrv A culture of £ coli is grown to mid-log phase m nutnent broth with shaking at 37 °C The log-phase culture is resuspended m 6 tubes of fresh 0 2μ-filtered tryptone broth, each contammg 4 mL of 2 x 10 ⁶ cfu/mL To each tube is added 4 mL of fresh 0 2μ-filtered tryptone broth containmg a 2X concentration of ampicillin (2 ,20, 200, 2000, 20000 μg/mL), or tryptone broth alone (control) The suspensions are mcubated for 0, 2, 4 and 6 hours and 2 mL of sample is removed at each time pomt To the 2 mL sample, 2 μL of 5 mM Dye 398 is added and the suspension is mcubated for 10 mmutes The distnbutions of the fluorescence intensities are analyzed by flow cytometry with 488 nm excitation and channel 1 (green) fluorescence emission detection Fluorescence mtensity is then plotted against the forward scatter of the bactena for each time of mcubation with ampicillin to determme the minimum inhibitory concentration (MIC) of ampicillin

Example 44 In situ assessment of neutroohil bactencidal activity

The differential permeability of Dye 397 for mammalian cells but not for live bactena is used to determme the viability of phagocytosed bactena Adherent cells, mcludmg neutrophils and macrophages, are purified from human peripheral blood E coli are grown to late log-phase in nutnent broth and opsomzed with rabbit anti-E coli IgG, washed mto stenle water to a density of 1 x 10 ⁷ cfu /mL, and stamed by addition of 1 μlJmL of a 1 mM DMSO stock solution of red bactena-permeant Dye 314 The bactena are stained for 15 mmutes and then washed extensively to remove all traces of extracellular dye One μL/mL of a 1 mM DMSO solution of Dye 397 is added to the phagocytes and the culture is mcubated for 15 mmutes The residual dye is rinsed off with medium and fresh medium containmg 1 μM Dye 397 is added The labeled bactena are added to the dye-loaded cells and the bactencidal activity of the phagocytes is mdicated

by an mcrease in the progression of green fluorescent stammg of the mtracellular bactena, as observed m a microscope equipped with a fluorescem long-pass filter set

Example 45 Determination of metabolic activity of bactena using flow cvtometrv Salmonella typhimurium are grown to mid-log phase m nutnent broth at 37 °C Bactena are washed twice m stenle E- pure water and 1 x 1 OVmL S typhimurium are inoculated mto 50 mL of tryptone medium of different strengths 100%, 10%, 1%, and 0% (pure water) After 4 hours growth at 37 °C each culture of bactena is concentrated by centrifugation at 10,000 x g for 10 mmutes, and permeabilized by subsequent resuspension in 70% isopropanol for 1 hour To an aliquot of the bactena cultured with 100% nutnent broth is added 20 μg of heat-inactivated RNase A, and the aliquot is mcubated at 37 °C for 60 mmutes All the bactenal samples are then washed twice by centrifugation and stamed with

Dye 1 1 14 at a final concentration of 5 μM for 30 minutes at room temperature

The bactenal samples are analyzed usmg a flow cytometer equipped with an argon laser The fluorescence detector is set to collect light around 530 nm The top signal cluster m Figure 8A represents loganthmically growing bactena (cultured m 100% broth) The somewhat lower signal cluster m Figure 8B is obtamed from a culture kept at 1 % nutnent broth for 3 hours The appearance of the resultmg scatter plot, relative to the 100% and 0% standards, gives a measure of the metabolic activity of the bactenal samples

Example 46 Assay of attachment of bactena to cell surfaces Madin-Darby Canine Kidney (MDCK) cells are cultured in 96-well plates to 70% confluence Growth medium is removed from the wells and replaced with 100 μL of stenle physiological saline (PS, 10 mM Na HEPES, 135 mM NaCl, 5 mM KC1, 1 mM MgCl ₂ , 2 mM CaCl ₂ , 5 mM glucose, pH 7 4) A culture of 100 mL oi Salmonella typhimurium bactena is grown by shaking at 200 φm 37 °C nutnent broth to mid log-phase The bactena are washed by centrifugation and resuspended m PS to a density of 2 x 10 ⁷ /mL Ten mL of the bactenal suspension is removed and killed by treatment with 70% isopropyl alcohol for 1 hour The killed bactena are then washed twice m PS and resuspended to the ongmal volume A parallel aliquot is washed twice in PS and resuspended m the same volume Five mL of each sample are mixed together and 10 μL of 5 mM Dye 1 1 14 is added The mixture is mcubated for 10 mmutes at room temperature Ten μL of Dye 314 is then added and the mixture is mcubated for an additional 20 mmutes The stamed bactenal suspension is then washed twice m PS and senally diluted 1 10 four tunes In tnp cate, 100 μL of each bactenal dilution, or PS alone, is added to wells m the 96-well plate containmg MDCK cells The plate is mcubated at 37 °C for 20 mmutes with agitation every 30 sec All wells are then gently washed three times with PS and filled with 150 μL of PS The green fluorescence of the wells is quantified m a multi-well fluorescence plate reader usmg excitation at 485 nm and emission at 520 nm, the red fluorescence is determined by excitation at 590 nm and emission at 620 nm The relative proportions of fluorescence are φmpared with standard wells containmg cells with different amounts of bactenal suspension

Example 47 Determination of cell membrane lntegntv using flow cvtometrv

Bactenal samples are suspended m water at a density of about 6 x 10 ⁶ bactena per mL of water Mammalian cells are suspended in HEPES-buffered salme at a density of about 1 x 10 ⁶ per mL The sample is stamed with Dye 1 1 14, a

universally cell-impermeant stain. Bacterial samples are stained with 5 μM Dye 11 14, mammalian cells are stained with 1 -5 μM Dye 1 1 14. After 30 minutes of incubation the sample is analyzed by flow cytometry on an instrument using the 488 nm line of the argon laser. Forward (low angle) light scatter is set at an amplification level suitable for the biological objects to be analyzed. The fluorescence detector is set to collect light around 530 nm. Generally, bacteria require logarithmic signal amplification, while mammalian cells can be analyzed with linear signal amplification. The relative amounts of viable and non-viable cells can be quantitated by comparison with the fluorescence and scatter characteristics of the control samples. The results of this experiment are shown in Figure 9 for a 1 : 1 mixture of living and dead bacteria. The uppermost cluster of signals corresponds to dead bacteria, while the lowermost cluster represents viable bacteria. The inset plot of Figure 9 shows the excellent correspondence between calculated and measured live/dead ratios. Similar results can be obtained for mammalian cells.

Example 48: Determination of the Cell Cycle Distribution of Eukaryotic Cells

A staining buffer is prepared that is lOO mM Tris/HCl set to pH 7.4; 154 mM NaCl, 1 mM CaCl ₂ , 0.5 mM MgCl ₂ , and

0.1 % Nonidet P-40. The buffer is supplemented with 500 nM of Dye 1 1 14 from a stock solution of 500 μM in dry DMSO. Human lymphocytes are centrifiiged to obtain a cell pellet, and resuspended gently in phosphate buffered saline

(PBS) to obtain a single cell suspension. This cell suspension is slowly injected into 4 volumes of absolute ethanol cooled using an ice-water bath, while the suspension is vortexed at maximal speed. The sample thus fixed in 80% ethanol (final concentration) is left for at least 1 hour in a freezer at -20 °C. The sample is centrifiiged to obtain a pellet, and the pellet is resuspended into at least 5 mL of PBS, and incubated for at least 15 minutes at room temperature. The sample is pelleted, and resuspended in PBS, and then 5 μg of RNase A is added per mL of cell suspension. The sample is mcubated for at least 30 minutes at 37 °C. The sample is pelleted and resuspended in the staining buffer, such that the suspension contains Ixl0 ⁵ to 5xl0 ⁵ cells per mL. After at least 15 minutes of staining at room temperature the sample is analysed on a flow cytometer equipped with an argon laser set at 100 mW output for the 488 nm line. The forward (low angle) light scatter signal amplification is set such that signals appear in the upper half of the signal detection range. The acquisition trigger logic of the instrument on the fluorescence detector is set such that it is collecting light around 530 nm (the "fluorescein detector"). The signal amplification rate of said detector is set such that signals from the sample under investigation emerge within the detection range. The distribution of signals from the 530 nm fluorescence detector is analyzed with a cell cycle distribution algorithm. As shown in Figure 10A, flow cytometric analysis typically shows the horizontal clouds of signals corresponding to cells in the G 1 , S and G2 compartments of the cell cycle. From this data is derived a histogram which shows the distribution of cells among the Gl , S and G2 compartments of the cell cycle (Figure 10B).

Alternatively, cell cycle distributions may be analyzed using a microscopic imaging system. In this case, cells grown on coverslips are rinsed twice with PBS at 37 °C, fixed in 3.7% formaldehyde in PBS at 37 °C for 10 minutes rinsed 3 or 4 times with PBS at room temperature, and permeabilized in acetone at -20 °C for 10 minutes. The fixed cells are then rehydrated in PBS at room temperature for 10 minutes and stained with 500 nM of Dye 1 1 14 in 2x saline-sodium citrate buffer for at least 15 minutes at room temperature. The coverslips are viewed and analyzed with an image analysis system dedicated to acquire signals in the fluorescein region of the visible light spectrum, and the distribution of signals is analyzed as above.

Example 49 Analysis of cell proliferation bv continuous bromodeoxyundine labeling and vaπable fluorescent labeling A 10 mM stock solution of 5-bromodeoxyuπdιne (BrUrd) m PBS is prepared, and filter stenlized A culture of human melanoma cells DMEM-F12 culture medium supplemented with 10% fetal bovme serum and 100 μM BrUrd is grown the dark at 37 °C for 50 hours The resulting cells are harvested by trypsinization, centrifiiged, washed once with PBS, and the pellet is resuspended a buffered solution The resulting suspension is supplemented with 1 2 μg of

Hoechst 33342 dye such that the suspension contains lxlO ⁵ to 5xl0 ⁵ cells per mL The sample is stamed at room temperature the dark for at least 15 mmutes To the stamed sample is added Dye 1 1 14 in an amount to produce a final concentration of 50 nM, and the sample is stamed an additional 15 mmutes at room temperature in the dark The sample is analysed on a flow cytometer equipped with two argon lasers, the first is set at 100 mW output for the 488 nm lme, and the second at 40 mW output for the U V line The forward (low angle) light scatter signal amplification is set such that signals from the 488 nm laser lme appear in the upper half of the signal detection range The acquisition tngger logic of the instrument on the fluorescence detector is set to collect light around 530 nm The signal amplification rate of the detector that is collecting light between 400 and 480 nm emergmg from the UV laser beam (the "Hoechst channel"), and the detector that is collecting light around 530 nm from the 488 nm excitation (the "1 1 14 channel") are set such that all signals from the sample under investigation emerge within the detection ranges of both detectors The distnbution of signals from the fluorescence detectors is analyzed usmg an appropπate software package

The typical results of the analysis are shown m Figure 1 1 Figure 1 1 A shows a bivaπate cytogram displaying signal distπbutions m the Hoechst channel (abscissa) and 1 1 14 channel (ordinate) The nghtmost cluster m the cytogram, labeled GO/G 1 , represents cells which have not entered cell cyclmg during the observation penod The signal trail moving left-upward from this cluster represents cells m the S phase of the first cell cycle At the end of this trail appear cells which amved m the G2 phase of the first cell cycle Cells which have undergone mitotic division appear to the left and downward from the G2 cluster (labeled as G 1 ') Figure 11 B shows the distnbution of cells among the cell cycle compartments alongside the Hoechst axis

It is to be understood that, while the foregomg mvention has been descnbed m detail by way of illustration and example, numerous modifications, substitutions, and alterations are possible without departing from the spint and scope of the mvention as descnbed in the followmg claims