Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
  • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
  • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
  • C09B67/0033—Blends of pigments; Mixtured crystals; Solid solutions
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

• the present invention relates to methods for generating molecules with a high fluorescence output from molecules with a lower or negligible fluorescence and which may be catalysed by enzymatic action.
• the invention relates to methods for achieving fluorescence detection of analytes by using enzymatic means to generate fluorescent reporter molecules.
• fluorescence as a detection modality in biological assays is widespread, and a diverse variety of procedures are available to generate fluorescence in assay conditions for detection by a wide range of techniques. These techniques include fluorescence microscopy, fluorescence immunoassay and flow cytometry.
• Among the methods used to generate a fluorescent signal are those which use an enzyme to convert a non-fluorescent substrate to a fluorescent product.
• the enzyme may be coupled to an assay component, for example to an antibody in an immunoassay or to a nucleic acid molecule in a nucleic acid hybridisation assay, and is typically used to generate a fluorescence signal from a non-fluorescent substrate.
• the fluorescence intensity of the product provides the assay signal, and correlates with the amount of analyte in the assay (e.g. an antigen in an immunoassay or a complementary nucleic acid sequence in a nucleic acid hybridisation assay). Examples of such enzyme based fluorescence method assays are described in "Applications of Fluorescence in Immunoassays", Chapter 9, pages 223-232, I. A.
• the enzyme may be generated by protein synthesis in the course of the assay.
• a gene usually termed a reporter gene, which is inserted into a cell or organism in which it does not occur naturally or is only found at low levels, in such a way that expression of the reporter gene is linked to the expression of a cellular gene of interest.
• Consequent processing of the enzyme's non-fluorescent substrate to a fluorescent product correlates with the expression of the reporter gene, and hence provides an indirect measure of the expression of the cellular gene of interest.
• Enzymes that are synthesised from reporter genes and currently used in fluorescent assays for the measurement of in vivo gene expression include ⁇ -galactosidase, alkaline phosphatase and ⁇ - lactamase.
• ⁇ -galactosidase is a bacterial enzyme and has been well characterised for use in such enzyme- reporter assays in combination with substrates which can be rendered fluorescent by enzyme activity.
• the detection of ⁇ -galactosidase expression is described in "Fluorescence Microscopy and Fluorescent Probes", pages 211-215, J. Slavik, Plenum Press, London, 1996.
• a non- fluorescent substrate, CMFDG is microinjected into cells expressing the enzyme where it is hydrolysed into a fluorescent form giving a measure of enzyme activity.
• Mammalian cells can also have some endogenous alkaline phosphatase enzyme activity again leading to a background of activation of a non-fluorescent substrate into its fluorescent form in in vivo assays based on alkaline phosphatase expression.
• the fluorescent substrate is, typically, fluorescein diphosphate.
• optimal alkaline phosphatase activity requires pH 9.8 which limits the use of this enzyme in in situ assays.
• a fluorescent substrate for ⁇ -lactamase has been documented (see US patent no. 5,955,604, Tsien et al.).
• the substrate described therein, CCF2 is suitable for use in a FRET based assay (as described below) in which the donor and acceptor molecules in the fluorescent substrate are separated by the enzyme activity of ⁇ -lactamase to generate a fluorescent signal from the donor molecule.
• fluorochromes used in these techniques are, typically, fluorescein and its derivatives (Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 6 Edition, 1996 or see www.probes.com).
• Fluorescein-based fluorochromes typically have excitation wavelengths in the UN to blue region of the spectrum and emission wavelengths in the blue to green region of the spectrum (i.e. excitation at approx. 488nm and emission at approx. 510nm). These characteristics confer certain restrictions on the use of these fluorochromes with biological materials as these wavelengths coincide with the excitation and emission ranges of a number of biological molecules. This can give high background fluorescence in biological assays with a resultant reduced sensitivity of detection.
• Dyes based on fluorescein have a number of other disadvantages, including their tendency to photobleach when illuminated by strong excitation sources. Furthermore, some products from enzyme substrates based on these fluorochromes are pH sensitive, which can lead to variation in fluorescence in different environments. Problems may arise using these fluorochromes in intracellular assays as excitation in the UN region, requiring high energy excitation at a short wavelength, may give rise to cell damage which can, in turn, lead to misleading results.
• a molecule which is the substrate for a non-ubiquitous enzyme and which has zero or low fluorescence in a first form and which, on reaction with the enzyme, yields an environmentally stable fluorescent product which can emit light over a broad range of the spectrum.
• the range of emission could be, for example in the range of 500-900 nm region of the spectrum.
• Cyanine dyes (sometimes referred to as "Cy dyeTM"), described, for example, in US Patent 5,268,486, are a series of biologically compatible fluorophores which are characterised by high fluorescence emission, environmental stability and a range of emission wavelengths extending into the near infra-red which can be selected by varying the internal molecular skeleton of the fluorophore.
• FRET Fluorescence Resonance Energy Transfer
• the principle has been used in the detection of binding events or cleavage reactions in assays which employ FRET.
• a fluorescent donor molecule and a fluorescent acceptor molecule are attached to a peptide substrate on either side of the peptide bond to be cleaved and at such a distance that energy transfer takes place.
• a peptide cleavage reaction will separate the donor and acceptor molecules and thus the fluorescence of the donor molecule will be restored.
• a fluorescent moiety is caused to be in close proximity with a "quencher” molecule such that the energy from the excited donor fluorophore is transferred to the quencher and dissipated as heat rather than fluorescence energy.
• quencher a fluorescent moiety
• residual fluorescence is minimised when the two components of the donor-quencher pair are in close proximity and a large change in signal can be obtained when they are separated.
• Cyanine dyes suitable for use as acceptor or "quencher” molecules in a FRET assay have been developed (see PCT/GB99/01746) by making certain modifications to cyanine dyes through introduction of chemical groups which have the effect of diminishing or abolishing the fluorescence of the molecule.
• One example of such a chemical modification is the introduction of-NO2 groups.
• Such quenched Cy dyes are referred to as Cy-Q dyes or "dark dyes”.
• reaction scheme 1 The bacterial enzymes termed nitroreductases have been shown to catalyse the general reaction set out below in reaction scheme 1 :
• Bacterial nitroreductases have been used, in anti-tumour therapy, to convert prodrug molecules into their corresponding cytotoxic forms (Anlezark et al 1995, Biochem-Pharmacol 50(5), 609- 18) by removing or reducing one c r more -NO- groups on the prodrug substrate.
• substrates include p-nitrobenzyloxycarbonyl derivatives of cytotoxic compounds.
• Selective killing of tumour cells can be achieved by targeting expression of the mtroreductase gene to tumour cells and administering the prodrug to the affected tissue (as described, for example, in US Patent No.5, 780,585).
• the prodrug substrate is converted to its cytotoxic form in the tumour cells expressing mtroreductase while the surrounding cells (which do not contain the nitroreductase) remain unaffected.
• Nitroreductases have also been used in bio-remediation processes for clearing nitroaromatic compounds which may create environmental or health hazards.
• US Patent No. 5,777,190 describes a catalytic method involving oxygen-sensitive nitroreductases for reducing nitroaromatic compounds which may be controlled, to prevent the reaction from progressing to completion, by the addition of oxygen.
• Suggested substrates include nitrobenzene, trinitrotoluene and orthochloronitrobenzene with preferred oxygen sensitive nitroreductase enzymes including ferredoxin NADP, xanthine oxidase and glutathione reductase.
• the present invention provides a method for increasing fluorescence of a modified cyanine dye comprising at least one NO 2 (nitro) group, for example, a Cy-Q dye.
• a modified cyanine dye comprising at least one NO 2 (nitro) group, for example, a Cy-Q dye.
• This can be achieved by enzymatic conversion of an NO 2 group in such a Cy-Q dye to a NHOH or NH by the action of a nitroreductase (NTR).
• NTR nitroreductase
• the fluorescence emission from the product of the Cy-Q/NTR reaction may occur across a wide range of wavelengths, typically 500-900nm, in contrast to existing reporters which emit only in the blue-green region of the spectrum. This emission at longer wavelengths is advantageous in avoiding background fluorescence and increasing sensitivity in biological systems.
• the fluorescence emission characteristics of the Cy-Q/NTR reaction product can be altered to suit the application by making changes to the internal structure of the Cy-Q molecule, without changing the extremities of the molecule, e.g. the NO 2 groups, that are involved in reaction with nitroreductase.
• fluorescent reporters compatible for use with other fluors in multiplex systems can be provided.
• the structure-defined emission characteristics of the Cy-Q make it suitable for inclusion in a paired fluorophore ratiometric reporter molecule where one member of the pair is a fluorescent dye and the second member is a Cy-Q dye. Nitroreductase action on the Cy-Q leads to a change in the ratio of fluorescence emission from the two fluors when excited and monitored at two different wavelengths.
• a ratiometric reporter molecule allows measurement of enzyme activity to be made independent of the concentration of the reporter molecule.
• the invention accordingly provides in a first aspect, a method for increasing the fluorescence of a dye molecule comprising at least one NO 2 group characterised by the reduction of said at least one NO 2 group to NHOH or NH .
• Suitable NO 2 -containing dyes include fluorescent dyes such as cyanine derivates, Cy-Q (described in PCT/GB99/017460), NO 2 -lanthanide chelates (described, for example, in Latra, M.J., Lumin. 1997, 75,149-169 and Blasse, G., J. Phys. Chem. 1998; 92; 2419-2422) and NO 2 - containing fluoresceins, pyrenes, rhodamines, coumarins or BODIPY dyes.
• fluorescent dyes such as cyanine derivates, Cy-Q (described in PCT/GB99/017460), NO 2 -lanthanide chelates (described, for example, in Latra, M.J., Lumin. 1997, 75,149-169 and Blasse, G., J. Phys. Chem. 1998; 92; 2419-2422) and NO 2 - containing fluoresceins, pyrene
• the dye molecule comprising at least one NO 2 group for use in the present invention is a modified cyanine dye compound having the Formula I:
• R 3 , R 4 , R 5 and R 6 are attached to the rings containing X and Y or, optionally, are attached to atoms of the Z 1 and Z 2 ring structures and n is an integer from 1-3;
• Z and Z each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms;
• R 3 , R 4 , R 5 , R 6 and R 7 are independently selected from the group consisting of hydrogen, substituted or unsubstituted C ⁇ -C 4 alkyl, OR 9 , COOR 9 , nitro, amino, acylamino, quaternary ammonium, phosphate, sulphonate and sulphate, where R 9 is substituted or unsubstituted and selected from H, CpC 4 alkyl, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary ammonium, nitro, primary amide, substituted amide, and groups reactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydryl groups.
• R 1 and R 2 are selected from d-Cio alkyl which may be unsubstituted or substituted;
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 comprises at least one nitro group which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent.
• the at least one nitro group comprised in the dyes of Formula I may be attached directly to the rings containing X and Y.
• a mono- or di-nitro-substituted benzyl group may be attached to the rings containing X and Y, which optionally may be further substituted with one or more nitro groups attached directly to the aromatic rings.
• R 1 and R 2 may be selected from d-C 10 alkyl which may be substituted with groups including NH 2 , OH, COOH, SO 3 H, PO 4 H, SH, polyethylene glycol and phenyl. Where phenyl is substituted, it may optionally be substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups.
• NO 2 -containing "dark dye” forms of any of the following Cy dyes are especially preferred.
• the excitation (Abs) and emission (Em) characteristics of the unmodified dye molecules are shown:
• the reduction of a NO 2 group is catalysed by an enzyme, which can be a nitroreductase and, preferably, a bacterial nitroreductase.
• cyanine-based dyes can be used in such an enzyme-substrate reaction at the same time as any of the conventional enzyme-substrate reactions which give a fluorescence readout in the blue/green region of the spectrum. Measurements can be made simultaneously using two different wavelengths; for example, fluorescein-based molecules could be detected at Abs 488/Em 510 whereas reduced cyanine-based molecules, such as those based on Cy5, could be detected at Abs 649/Em 670. This would allow multiplexing i.e. measuring a number of different in vitro or in vivo effects simultaneously.
• non-fluorescent dyes i.e. those dyes which have an intrinsic low efficiency for converting absorbed incident light into fluorescence.
• the effectiveness of a non-fluorescent dye as a nitroreductase substrate is the extent to which it can convert incident light to fluorescence after a reduction reaction.
• An increase in fluorescence can be measured relative to a control sample comprising an NO 2 - containing cyanine dye molecule substrate in the absence of a nitroreductase enzyme.
• a method for detecting nitroreductase enzyme activity in a composition comprising: a) mixing said composition with a dye compound comprising at least one NO group under conditions to promote nitroreductase activity; and b) measuring an increase in fluorescence wherein said increase is a measure of the amount of nitroreductase activity.
• the dye compound is a cyanine dye compound comprising at least one NO group.
• the cyanine dye compound is a compound of Formula I as hereinbefore described.
• the composition comprises a cell or cell extract.
• a cell or cell extract can be prepared from a cell, using standard methods known to those skilled in the art (Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press 1989), prior to measuring fluorescence.
• Typical conditions for nitroreductase activity comprise incubation of the composition and a cyanine dye molecule comprising at least one NO group at approximately 37°C in the presence of NADH and FMN.
• a method of detecting analytes comprising: a) providing a nitroreductase enzyme coupled to an assay reagent under conditions where the amount of activity of the enzyme is proportional to the amount of analyte in the assay; b) providing a dye compound comprising at least one NO 2 group; and c) measuring an increase in fluorescence as a measure of the amount of nitroreductase activity.
• an assay method which comprises: a) binding a first component of a specific binding pair to a surface; b) adding a second component of the specific binding pair under conditions to promote binding between the components, said second component being labelled with a nitroreductase enzyme; c) adding a dye compound comprising at least one NO group under conditions suitable for nitroreductase activity; and d) detecting binding of the second component to the first component by measuring an increase in fluorescence as a measure of bound nitroreductase activity.
• the dye compound is a cyanine dye compound comprising at least one NO group.
• the dye compound employed in the third or fourth aspects is a cyanine dye compound of Formula I as hereinbefore described.
• said specific binding pair is selected from the group consisting of antibodies/antigens, lectins/glycoproteins, biotin/streptavidin, hormone/receptor, enzyme/substrate, DNA/DNA, DNA/RNA, DNA/binding protein or engineered binding partner.
• an in vitro assay method for the detection of antibody binding may be configured as follows.
• An antibody specific for an antigen of interest may be labelled by covalently linking it to an enzymatically active nitroreductase.
• Said labelled antibody can then be introduced into the test sample containing the antigen under binding conditions. After washing to remove any unbound antibody, the amount of antibody bound is detected by incubating the sample with the non-fluorescent cyanine dye substrate under conditions for nitroreductase activity and measuring an increase in fluorescence. The amount of fluorescence detected will be proportional to the amount of nitroreductase-labelled antibody that has bound to the analyte.
• either of the pair of target and probe nucleic acid is bound to a membrane or surface.
• the unbound partner is labelled with nitroreductase and incubated under hybridising conditions with the bound nucleic acid.
• Unbound, labelled nucleic acid is washed off and the amount of bound, labelled nucleic acid is measured by incubating the membrane or surface with a non-fluorescent cyanine dye under conditions suitable for nitroreductase activity. The amount of increase in fluorescence gives a measure of the amount of bound labelled DNA.
• Coupling may be achieved by direct means, for example by use of a suitable bifunctional crosslinking agent (e.g. N-[ ⁇ - Maleimidopropionic acid]hydrazine, Pierce) to covalently link the enzyme and binding partner.
• a suitable bifunctional crosslinking agent e.g. N-[ ⁇ - Maleimidopropionic acid]hydrazine, Pierce
• coupling may be achieved by indirect means, for example by separately biotinylating the enzyme and the binding partner using a chemically reactive biotin derivative, (e.g. N-hydroxysuccinimido-biotin, Pierce) and subsequently coupling the molecules through a streptavidin bridging molecule.
• Cell based assays are increasingly attractive over in vitro biochemical assays for use in high throughput screening (HTS). This is because cell based assays require minimal manipulation and the readouts can be examined in a biological context that more faithfully mimics the normal physiological situation.
• Such in vivo assays require an ability to measure a cellular process and a means to measure its output. For example, a change in the pattern of transcription of a number of genes can be induced by cellular signals triggered, for example, by the interaction of an agonist with its cell surface receptor or by internal cellular events such as DNA damage. The induced changes in transcription can be identified by fusing a reporter gene to a promoter region which is known to be responsive to the specific activation signal.
• an assay method which comprises: a) contacting a host cell with a dye compound comprising at least one NO 2 group, wherein said host cell has been transfected with a nucleic acid molecule comprising expression control sequences operably linked to a sequence encoding a nitroreductase; and b) measuring an increase in fluorescence as a measure of nitroreductase gene expression.
• the dye compound is a cyanine dye compound comprising at least one NO group.
• the cyanine dye is a compound of Formula I as hereinbefore described.
• reporter gene is chosen to allow the product of the gene to be measurable in the presence of other cellular proteins and is introduced into the cell under the control of a chosen regulatory sequence which is responsive to changes in gene expression in the host cell.
• Typical regulatory sequences include those responsive to hormones, second messengers and other cellular control and signalling factors.
• agonist binding to seven transmembrane receptors is known to modulate promoter elements including the cAMP responsive element, NFAT, SRE and API;
• MAP kinase activation leads to modulation of SRE leading to Fos and Jun transcription; DNA damage leads to activation of transcription of DNA repair enzymes and the tumour suppressor gene p53.
• the reporter gene can be used to assay the effect of added agents or cellular processes involving the chosen regulatory sequence under study.
• the nitroreductase gene may be isolated by common methods, for example by amplification from a cDNA library by use of the polymerase chain reaction (PCR) (Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press 1989 pp 14.5-14.20). Once isolated, the nitroreductase gene may be inserted into a vector suitable for use with mammalian promoters (Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press 1989 pp 16.56-16.57) in conjunction with and under the control of the gene regulatory sequence under study.
• PCR polymerase chain reaction
• the vector containing the nitroreductase reporter and associated regulatory sequences may then be introduced into the host cell by transfection using well known techniques, for example by use of DEAE-Dextran or Calcium Phosphate (Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press 1989 pp 16.30-16.46). Other suitable techniques will be well known to those skilled in the art.
• Nitroreductase has been shown to be retained in cells when expressed in this manner (see Bridgewater et al. Eur. J. Cancer 31a, 2362-70).
• the cyanine dye molecule comprising at least one NO group is permeable to cells.
• at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 of the cyanine dye molecule of Formula I comprises a cell membrane permeabilising group.
• Membrane permeant compounds can be generated by masking hydrophilic groups to provide more hydrophobic compounds. The masking groups can be designed to be cleaved from the fluorogenic substrate within the cell to generate the derived substrate intracellularly. Because the substrate is more hydrophilic than the membrane permeant derivative it is then trapped in the cell.
• Suitable cell membrane permeabilising groups may be selected from acetoxymethyl ester which is readily cleaved by endogenous mammalian intracellular esterases (Jansen, A.B.A. and Russell, T.J., J.Chem Soc. 2127-2132 (1965) and Daehne W. et al. J.Med-.Chem. 13, 697-612 (1970)) and pivaloyl ester (Madhu et al., J. Ocul. Pharmacol. Ther. 1998, 14, 5, pp 389-399) although other suitable groups including delivery molecules (such as delivery peptides) will be recognised by those skilled in the art.
• an NO 2 -containing compound in accordance with Formula I wherein said compound has been modified so as to be capable of entering a cell. Accordingly, in a particularly preferred embodiment, there is provided an NO 2 -containing compound selected from compounds of Formula Ilia or Formula nib.
• cells transfected with the nitroreductase reporter are incubated with the test agent, followed by addition of a cell-permeant cyanine dye substrate, such as a cyanine dye molecule comprising at least one NO group.
• a cell-permeant cyanine dye substrate such as a cyanine dye molecule comprising at least one NO group.
• the fluorescence emission from the cells is measured at a wavelength appropriate for the chosen cyanine dye. For example, for the compound Cy-Q F (shown in Figure la), fluorescence emission would be monitored at 690nm with excitation at 650nm.
• Measurement of fluorescence may be readily achieved by use of a range of detection instruments including fluorescence microscopes (e.g. LSM 410, Zeiss), microplate readers (e.g. CytoFluor 4000, Perkin Elmer), CCD imaging systems (e.g. LEADseekerTM, Amersham Pharmacia Biotech) and Flow Cytometers (e.g. FACScalibur, Becton Dickinson).
• fluorescence microscopes e.g. LSM 410, Zeiss
• microplate readers e.g. CytoFluor 4000, Perkin Elmer
• CCD imaging systems e.g. LEADseekerTM, Amersham Pharmacia Biotech
• Flow Cytometers e.g. FACScalibur, Becton Dickinson.
• the measured fluorescence is compared with fluorescence from control cells not exposed to the test agent and the effects, if any, of the test agent on gene expression modulated through the regulatory sequence is determined from the ratio of fluorescence in the test cells to the fluorescence in the control cells.
• a cell extract can be prepared using conventional methods.
• an assay method which comprises: a) contacting a host cell extract with a dye compound comprising at least one NO 2 group wherein said host cell has been transfected with a nucleic acid molecule comprising expression control sequences operably linked to a sequence encoding a nitroreductase; and b) measuring an increase in fluorescence as a measure of nitroreductase gene expression.
• the dye compound is a cyanine dye compound comprising at least one NO group.
• the cyanine dye is a compound of Formula I as hereinbefore described.
• increased fluorescence of the cyanine dye molecule is identified by analysis of fluorescence emission in the range 500 to 900 nm, preferably 550-780nm, and, most preferably 665-725 nm.
• kits for a reporter system comprising a means for expressing a nitroreductase enzyme and a dye molecule comprising at least one NO 2 group.
• Suitable means for expressing a nitroreductase enzyme include an expression plasmid or other expression construct. Methods for preparing such expression constructs are well known to those skilled in the art.
• a kit for detecting the presence of one component of a specific binding pair comprising a nitroreductase enzyme coupled to the other component of said specific binding pair and a dye molecule comprising at least one NO 2 group.
• the dye molecule is a cyanine dye molecule comprising at least one NO 2 .
• the cyanine dye is a compound of Formula I as hereinbefore described.
• D] is a detectable fluorophore
• D 2 is a cyanine dye molecule having Formula (I).
• L is a linker group
• Dj and D 2 are selected such that excitation of the cassette is at two different wavelengths, ⁇ l and ⁇ 2, where the wavelengths are chosen to be suitable to elicit fluorescence emission from the fluorophore Di (at wavelength ⁇ 3) and the fluorophore corresponding to D 2 (at wavelength ⁇ 4), and from which D 2 is derived, yet lacking the at least one ⁇ O 2 group.
• D 2 is selected from Cy3Q and Cy5Q.
• L is a cleavable linker, for example, chemically cleavable, photocleavable (e.g. nitrobenzylalcohol) or enzymatically cleavable (e.g. ester, amide, phosphodiester, azo) by enzymes such as proteases.
• cleavable linker for example, chemically cleavable, photocleavable (e.g. nitrobenzylalcohol) or enzymatically cleavable (e.g. ester, amide, phosphodiester, azo) by enzymes such as proteases.
• Suitable methods for cleaving such a linker are well known and described, for example, in Gerard Marriott et al., Preparation and photoactivation of caged fluorophores and caged proteins using a new cross-linking reagent, Bioconjugate Chemistry; (1998); 9(2); 143-151 and WO 00/75358.
• D] and D 2 may be in a FRET arrangement.
• the compound of Formula IV is rendered cell or membrane permeable.
• Di 8-hydroxy-pyrene-l,3,6-trisulfonic acid (Cascade Blue TM), D is Cy5Q and L is an ester linkage.
• Dj and D 2 are arranged in a FRET arrangement such that the fluorescence of Di is quenched by D 2 .
• the linker, L is cleaved thus releasing Di from the energy transfer arrangement and, when excited at wavelength ⁇ l, fluorescence emission at wavelength ⁇ 3 can be detected.
• the compound of Formula IV may be used in an assay method in accordance with any one of the third, fourth, fifth, sixth or eighth aspects of the invention.
• Figure la shows the chemical structure of the compound of Formula II, termed Cy-Q F.
• Figure lb shows a fluorescence emission spectrum of Cy-Q F incubated for various times in the presence of E.coli B nitroreductase.
• FIG. 1 shows the chemical structure of the compound of Formula III, termed Cy-Q G.
• Figure 2b shows a fluorescence emission spectrum of Cy-Q G incubated for various times in the presence of E.coli B nitroreductase.
• Figure 3a shows HPLC analysis of Cy-Q G.
• Figure 3b shows HPLC analysis of Cy-Q G treated with nitroreductase.
• Figure 4a shows MALDI-TOF Mass Spectrometry analysis of Cy-Q G.
• Figure 4b shows MALDI-TOF Mass Spectrometry analysis of Cy-Q G treated with nitroreductase.
• Figure 5 shows infra-red absorbtion spectroscopy of Cy-Q G and Cy-Q G treated with nitroreductase.
• Figure 6 shows the chemical structure of Cy5Q.
• Figure 7 shows fluorescence emission different concentration of Cy5Q in the presence nitroreductase.
• Figure 8 shows the results of an in vitro binding assay with nitroreductase.
• Figure 9 shows detection of nitroreductase activity in cell lysates from transfected and control cells by detecting Cy5 fluorescence.
• Figure 10 shows Cy3 fluorescence in cells expressing nitroreductase and control cells.
• Figure 11 shows Cy5 fluorescence in cells expressing nitroreductase and control cells.
• Figure 12 shows measurement of cellular nitroreductase activity by flow cytometry.
• Figure 16 shows relative fluorescence of Cy5Q-cascade blue cassette in the presence of absence of lysate/NTR.
• a reaction mixture was prepared containing 0.8mg/ml E.coli B nitroreductase, ImM NADH, 0.4 ⁇ m FMN and 0.05mM Cyanine-Q F (Cy-Q F, Figure la) in phosphate buffered saline on ice. A sample was removed immediately for analysis by fluorescence spectroscopy, and further samples were removed for analysis after incubation of the reaction mixture at 37°C for 1, 2, and 5 hours.
• a reaction mixture was prepared containing 0.8mg/ml E.coli B nitroreductase, ImM NADH, 0.4 ⁇ m FMN, and 0.05mM Cyanine-Q F (Cy-Q G, Figure 2a) in phosphate buffered saline on ice. A sample was removed immediately for analysis by fluorescence spectroscopy, and further samples were removed for analysis after incubation of the reaction mixture at 37°C for 1, 2, and 5 hours.
• E.coli B nitroreductase 500ng was incubated in the presence of increasing concentrations of
• Nitroreductase 400ug, 16.7nmol was labelled with 334nmol Sulpho-NHS-biotin (Pierce) in
• biotin Sigma
• concentrations of biotin (Sigma) from 0-lOOnmol/well were added to duplicate wells of a streptavidin-coated microtitre plate (Pierce) in lOOul PBS pH7.4, followed by lOOul of PBS containing 0.5ug biotinylated nitroreductase, and the plate incubated for 1 hour at room temperature.
• the E.coli nitroreductase B gene was cloned into the p-Target mammalian expression vector
• cell lysates were prepared from transfected and non- transfected control cells by sonication in phosphate buffered saline. Nitroreductase activity was determined by addition of 2 ⁇ M Cy5Q and incubating at room temperature for 90 minutes followed by measurement of fluorescence in a CytoStar (PerSeptive Biosystems) plate reader ( Figure 9) using 610/20nm excitation and 670/40nm emission filters. Results showed a strong increase in fluorescence in lysate samples from nitroreductase expressing cells which was proportional to the amount of cell lysates assayed.
• CytoStar PerSeptive Biosystems
• Methyl iodide (3ml, 48.19mmol) was added to a solution of 2,3,3-trimethyl-3H-indol-5-yl-acetic acid (2.5g, 11.52mmol) in sulfolan (15ml). The reaction was heated at 48°C for 18 hours, then cooled to room temperature. The crude reaction mixture was added dropwise to an excess of diethyl ether and the precipitate was collected by filtration and dried in vacuo to obtain the product as a beige solid (3.27g, 79% yield).
• 3,5-Dinitrobenzyl chloride (12.46g, 57.5mmol) was added to a solution of 2,3,3-trimethyl-3H- indol-5-yl-acetic acid (2.5g, 11.5mmol) with sodium bromide (5.92g, 57.5mmol) in sulfolan (10ml). The reaction was heated at 100°C for 20hrs, then cooled to room temperature. The crude reaction mixture was added dropwise to an excess of ethyl acetate and the dark brown solid was filtered OFF and dissolved in dimethylsulfoxide before purification by reverse phase chromatography. The product was isolated as beige solid (54% yield).
• Cy3Qee (Formula Ilia) and Cy5Qee (Formula Illb) were evaluated as cell permeable fluorescence substrates for nitroreductase measurement in living cells.
• Nitroreductase expressing cells and control cells were cultured at 10,000 cells/well in 96 well plates in tissue culture medium containing 10% foetal calf serum and incubated at 37°C in the presence of lO ⁇ M Cy3Qee or 30 ⁇ M Cy5Qee.
• Fluorescence measurements were made using a CytoStar (PerSeptive Biosystems) plate reader using 530/25nm excitation and 580/50nm emission filters for Cy3Qee and 610/20nm excitation and 670/40nm emission filters for Cy5Qee.
• Nitroreductase expressing cells and control cells were incubated for 2 hours at 37°C in tissue culture media containing 30 ⁇ M Cy3Qee. Following incubation cells were washed with phosphate buffered saline and trypsinised to produce cell suspensions for flow cytometry.
• Cy5Q mono free acid potassium salt obtained from Amersham Pharmacia Biotech Ltd
• Cascade blue ® (Molecular Probes)
• 8-hydroxypyrene-l,3,6-trisulfonic acid sodium salt (8.8mg, 0.018 mmol) (Fluka)
• N, N diisopropylcarbodiimide (lO ⁇ l, 0.065mmol)
• 1- hydroxybenzotriazole lmg, 0.007 mmol
• 4-(dimethylamino)pyridine 0.0.9mg, 0.007 mmol
• activated molecular sieves powder 250mg
• Cy5Q-Cascade blue cassette (0.8mM in acetate buffer pH5.0) was diluted to 8 ⁇ M in PBS pH7.4 containing ImM NADH.
• a cell lysate was prepared from 2 x10 SKOV cells by re-suspension of scraped cells in 5ml PBS at 4°C and repeated passage through a 25 gauge syringe needle. Aliquots (1ml) of Cy5Q-Cascade blue were incubated with 500 ⁇ l of cell lysate or PBS for 20 minutes at 37°C. Following incubation lOO ⁇ l of a lng/ ⁇ l solution of E.coli B nitroreductase was added to one sample and incubation continued for 5 minutes.

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