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  • C07C245/08—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
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  • C07C245/06—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings
  • C07C245/10—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings being part of condensed ring systems
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  • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D241/16—Halogen atoms; Nitro radicals
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  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
• • G—PHYSICS
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  • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

• This invention relates generally to the field of molecular biology and more particularly, to methods and reagents of biomolecular detection and fluorescence-based assays.
• Fluorescence resonance energy transfer is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity, conformation, and bond formation and cleavage. FRET has been applied in detection of labelled biomolecules to important areas of genomics: identification of single-nucleotide polymorphisms and other allelic variants, protein-protein interactions, and genome-wide analysis of regulatory sequences. Genetic information is being rapidly gleaned by automated sequencing of genomes from all kinds of organisms.
• Fluorescent quenching acceptors e.g. TAMRA (tetramethylrhodamine) have been employed in FRET assays, but have at least two significant limitations: background fluorescence and the preclusion of detection of reporter fluorescence at the acceptor fluorescence emission.
• background fluorescence the emission maxima at about 582 mn of TAMRA-labelled oligonucleotide probes hinders the use of reporter dyes with comparable emission maxima.
• the present invention provides bis-diazo,triaryl I and aryldiazo-N-arylphenazonium II compounds which are useful as quencher labels when attached to biomolecules such as polynucleotides, nucleosides, nucleotides, carbohydrates, and polypeptides.
• the fluorescence quencher compositions have structure III:
• Y is N or CR, where R is H, C 1 -C 6 alkyl or C 5 -C 14 aryl.
• L 1 , L 2 , and L 3 are each, independently, a bond, or a linker such as C 1 -C 12 alkylidyl, C 1 -C 12 alkoxydiyl, C 1 -C 12 alkylaminodiyl, C 1 -C 12 alkylamidediyl, C 5 -C 14 aryldiyl, and 1-20 ethyleneoxy units.
• X is a biomolecule such as an amino acid, a polypeptide, a nucleoside, a nucleotide, a polynucleotide, or protected form thereof; or X may be an acid-labile protecting group which can be removed to form a reactive linking group which can form an attachment to a biomolecule.
• Z is a reactive linking group to form attachments to biomolecules and labels, a solid support, or a label.
• Q is a bis-diazo,triaryl quencher moiety I or aryldiazo-N-arylphenazonium quencher moiety II.
• One of the aryl carbons of a quencher moiety, Q, is attached to linker L 1 .
• At least one aryl carbon of the quencher moiety is substituted with an electron-withdrawing group, and at least one aryl carbon of a quencher moiety is substituted with an electron-donating group.
• the electron-withdrawing group may be on the same or different aryl ring as the electron-donating group.
• compositions comprising nucleosides, nucleotides, polynucleotides, or polypeptides labelled with the bis-diazo,triaryl and aryldiazo-N-arylphenazonium quencher moieties.
• the quencher labelled biomolecules may further contain fluorescent reporter moieties which form energy transfer pairs.
• polynucleotides and polypeptides with the bis-diazo,triaryl and aryldiazo-N-arylphenazonium quencher moieties of the invention.
• polynucleotides can be labelled at the 3′ terminus with fluorescence quencher solid support compositions.
• Polynucleotides can be labelled at the 5′ terminus with fluorescence quencher phosphoramidite compositions.
• Another aspect of the invention are methods of primer extension where a polynucleotide primer is annealed to a target polynucleotide and extended by polymerase-mediated incorporation of a nucleotide 5′-triphosphate.
• the primer or a nucleotide 5′-triphosphate may be labelled with a quencher moiety of the invention.
• the 3′ terminus of the quencher labelled nucleotide may be modified to terminate primer extension.
• the resulting extension fragments may be separated and analyzed.
• Embodiments of primer extension methods include nucleic acid amplification.
• a target polynucleotide may be amplified by the polymerase chain reaction, or other nucleic acid amplification method, with nucleotide 5′-triphosphates, a polymerase, and two or more primers.
• a primer is labelled with a quencher moiety of the invention.
• a nucleotide 5′-triphosphate is labelled with a quencher moiety of the invention.
• a detectable probe may be labelled with a fluorescent dye and a quencher moiety. Hybridization to the target polynucleotide may be monitored by FRET and used to detect the target sequence.
• the probe may be cleaved by nuclease activity of an enzyme during nucleic acid amplification. Cleavage may generate a detectable signal and used to monitor and detect nucleic acid hybridization and amplification.
• the primers and probes may be further labelled with hybridization-stabilizing moieties, such as minor groove binders.
• primer extension is a method of fragment analysis where polynucleotide fragments are formed by polymerase-directed primer extension of a primer.
• the primer can be labelled with a quencher moiety of the invention.
• the fragments are resolved, i.e. separated, and detected.
• Another aspect of the invention is a method of oligonucleotide ligation.
• a probe labelled with a quencher moiety is hybridized to a complementary sequence, and adjacent to another probe labelled with a fluorescent reporter.
• Ligation may be performed with a ligase, generating a polynucleotide bearing the quencher moiety and the fluorescent reporter which interact to give a detectable fluorescent change.
• Another aspect of the invention includes a method for hybridization detection.
• a probe labelled with a fluorescent dye and a quencher moiety of the invention is annealed to a target polynucleotide sequence and a signal is detected from the fluorescent dye.
• the invention further includes kits of reagents for performing the methods and uses detailed herein.
• the kits may contain the fluorescence quencher compositions, biomolecules labelled with the quencher moieties, and/or other reagents.
• Biomolecule means an amino acid, a polypeptide, a nucleoside, a nucleotide, a polynucleotide, a carbohydrate, a vitamin, a hormone, and any other compound produced by an organism.
• Nucleobase means any nitrogen-containing heterocyclic moiety capable of forming Watson-Crick hydrogen bonds in pairing with a complementary nucleobase or nucleobase analog, e.g. a purine, a 7-deazapurine, or a pyrimidine.
• Typical nucleobases are the naturally occurring nucleobases adenine, guanine, cytosine, uracil, thymine, and analogs of the naturally occurring nucleobases, e.g.
• Nucleoside refers to a compound consisting of a nucleobase linked to the C-1′ carbon of a sugar, such as ribose, arabinose, xylose, and pyranose.
• the sugar may be substituted or unsubstituted.
• Substituted ribose sugars include, but are not limited to, those riboses in which one or more of the carbon atoms, for example the 2′-carbon atom, is substituted with one or more of the same or different Cl, F, —R, —OR, —NR 2 or halogen groups, where each R is independently H, C 1 -C 6 alkyl or C 5 -C 14 aryl.
• Ribose examples include ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose, 2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g. 2′-O-methyl, 4′- ⁇ -anomeric nucleotides, 1′- ⁇ -anomeric nucleotides, 2′-4′- and 3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications (WO 98/22489; WO 98/39352; WO 99/14226).
• Exemplary LNA sugar analogs within a polynucleotide include the structures:
• B is any nucleobase.
• Modifications at the 2′- or 3′-position of ribose include hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo.
• Nucleosides and nucleotides include the natural D optical isomer, as well as the L optical isomer forms (Garbesi (1993) Nucl. Acids Res. 21:4159-65; Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata, (1993) Nucleic Acids Symposium Ser. No.
• nucleobase is purine, e.g. A or G
• the ribose sugar is attached to the N 9 -position of the nucleobase.
• nucleobase is pyrimidine, e.g. C, T or U
• the pentose sugar is attached to the N 1 -position of the nucleobase ( Komberg and Baker, (1992) DNA Replication, 2 nd Ed., Freeman, San Francisco, Calif.).
• Nucleotide refers to a phosphate ester of a nucleoside, as a monomer unit or within a nucleic acid.
• Nucleotide 5′-triphosphate refers to a nucleotide with a triphosphate ester group at the 5′ position, and are sometimes denoted as “NTP”, or “dNTP” and “ddNTP” to particularly point out the structural features of the ribose sugar.
• the triphosphate ester group may include sulfur substitutions for the various oxygens, e.g. ⁇ -thio-nucleotide 5′-triphosphates.
• polynucleotide and “oligonucleotide” are used interchangeably and mean single-stranded and double-stranded polymers of nucleotide monomers, including 2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, or internucleotide analogs, and associated counter ions, e.g., H + , NH 4 + , trialkylammonium, Mg 2+ , Na + and the like.
• DNA 2′-deoxyribonucleotides
• RNA ribonucleotides linked by internucleotide phosphodiester bond linkages
• counter ions e.g., H + , NH 4 + , trialkylammonium, Mg 2+ , Na + and the like.
• a polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Polynucleotides may be comprised of nucleobase and sugar analogs. Polynucleotides typically range in size from a few monomeric units, e.g. 5-40 when they are more commonly frequently referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units.
• nucleotides are in 5′ to 3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted.
• Internucleotide analog means a phosphate ester analog or a non-phosphate analog of a polynucleotide.
• Phosphate ester analogs include: (i) C 1 -C 4 alkylphosphonate, e.g. methylphosphonate; (ii) phosphoramidate; (iii) C 1 -C 6 alkyl-phosphotriester; (iv) phosphorothioate; and (v) phosphorodithioate.
• Non-phosphate analogs include compounds wherein the sugar/phosphate moieties are replaced by an amide linkage, such as a 2-aminoethylglycine unit, commonly referred to as PNA (Buchardt, WO 92/20702; Nielsen (1991) Science 254:1497-1500).
• PNA 2-aminoethylglycine unit
• Polypeptide refers to a polymer including proteins, synthetic peptides, antibodies, peptide analogs, and peptidomimetics in which the monomers are amino acids and are joined together through amide bonds.
• amino acids are ⁇ -amino acids
• either the L-optical isomer or the D-optical isomer can be used.
• unnatural amino acids for example, valanine, phenylglycine and homoarginine are also included. Commonly encountered amino acids that are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L- optical isomer.
• other peptidomimetics are also useful in the present invention.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner choices can be found in many art recognized references.
• Attachment site refers to a site on a moiety or a molecule, e.g. a quencher, a fluorescent dye, a polynucleotide, or a PNA, to which is covalently attached, or capable of being covalently attached, a linker or another moiety.
• Linker refers to a chemical moiety in a molecule comprising a covalent bond or a chain of atoms that covalently attaches one moiety or molecule to another, e.g. a quencher to a polynucleotide.
• a “cleavable linker” is a linker which has one or more covalent bonds which may be broken by the result of a reaction or condition. For example, an ester in a molecule is a linker that may be cleaved by a reagent, e.g. sodium hydroxide, resulting in a carboxylate-containing fragment and a hydroxyl-containing product
• Reactive linking group refers to a chemically reactive substituent or moiety, e.g. a nucleophile or electrophile, on a molecule which is capable of reacting with another molecule to form a covalent bond.
• Reactive linking groups include active esters, which are commonly used for coupling with amine groups. For example, N-hydroxysuccinimide (NHS) esters have selectivity toward aliphatic amines to form aliphatic amide products which are very stable. Their reaction rate with aromatic amines, alcohols, phenols (tyrosine), and histidine is relatively low.
• NHS esters Reaction of NHS esters with amines under nonaqueous conditions is facile, so they are useful for derivatization of small peptides and other low molecular weight biomolecules.
• NHS esters are available with sulfonate groups that have improved water solubility.
• Substituted refers to a molecule wherein one or more hydrogen atoms are replaced with one or more non-hydrogen atoms, functional groups or moieties.
• an unsubstituted nitrogen is —NH 2
• a substituted nitrogen is —NHCH 3 .
• substituents include but are not limited to halo, e.g., fluorine and chlorine, C 1 -C 8 alkyl, sulfate, sulfonate, sulfone, amino, ammonium, amido, nitrile, nitro, alkoxy (—OR where R is C 1 -C 12 alkyl), phenoxy, aromatic, phenyl, polycyclic aromatic, heterocycle, water-solubilizing group, and linking moiety.
• halo e.g., fluorine and chlorine
• C 1 -C 8 alkyl sulfate, sulfonate, sulfone
• Alkyl means a saturated or unsaturated, branched, straight-chain, branched, cyclic, or substituted hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene, or alkyne.
• Typical alkyl groups consist of 1-12 saturated and/or unsaturated carbons, including, but not limited to, methyl, ethyl, cyanoethyl, isopropyl, butyl, and the like.
• Alkyldiyl means a saturated or unsaturated, branched, straight chain, cyclic, or substituted hydrocarbon radical of 1-12 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane, alkene or alkyne.
• Typical alkyldiyl radicals include, but are not limited to, 1,2-ethyldiyl (—CH 2 CH 2 —), 1,3-propyldiyl (—CH 2 CH 2 CH 2 —), 1,4-butyldiyl (—CH 2 CH 2 CH 2 CH 2 —), and the like.
• Alkoxydiyl means an alkoxyl group having two monovalent radical centers derived by the removal of a hydrogen atom from the oxygen and a second radical derived by the removal of a hydrogen atom from a carbon atom.
• Typical alkoxydiyl radicals include, but are not limited to, methoxydiyl (—OCH 2 —) and 1,2-ethoxydiyl or ethyleneoxy (—OCH 2 CH 2 —).
• Alkylaminodiyl means an alkylamino group having two monovalent radical centers derived by the removal of a hydrogen atom from the nitrogen and a second radical derived by the removal of a hydrogen atom from a carbon atom.
• alkylaminodiyl radicals include, but are not limited to —NHCH 2 —, —NHCH 2 CH 2 —, and —NHCH 2 CH 2 CH 2 —.
• Alkylamidediyl means an alkylamide group having two monovalent radical centers derived by the removal of a hydrogen atom from the nitrogen and a second. radical derived by the removal of a hydrogen atom from a carbon atom.
• Typical alkylamidediyl radicals include, but are not limited to —NHC(O)CH 2 —, —NHC(O)CH 2 CH 2 —, and —NHC(O)CH 2 CH 2 CH 2 —.
• Aryl means a monovalent aromatic hydrocarbon radical of 5-14 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
• Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like, including substituted aryl groups.
• Aryl carbon means any carbon atom part of an aromatic ring system.
• Aryldiyl means an unsaturated cyclic or polycyclic hydrocarbon radical of 5-14 carbon atoms having a conjugated resonance electron system and at least two monovalent radical centers derived by the removal of two hydrogen atoms from two different carbon atoms of a parent aryl compound, including substituted aryldiyl groups.
• Substituted alkyl mean alkyl, alkyldiyl, aryl and aryldiyl respectively, in which one or more hydrogen atoms are each independently replaced with another substituent.
• Typical substituents include, but are not limited to, F, Cl, Br, I, R, OH, —OR, —SR, SH, NH 2 , NMR, NR 2 , — + NR 3 , —N ⁇ NR 2 , —CX 3 , —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO 2 , —N 2 + , —N 3 , —NHC(O)R, —C(O)R, —C(O)NR 2 —S(O) 2 O ⁇ , —S(O) 2 R, —OS(O) 2 OR, —S(O) 2 NR, —S(O)R, —OP(O)(OR) 2 , —P(O)(OR) 2 , —P(O)(O ⁇ ) 2 , —P(O)(OH) 2 , —C(O)R, —C
• Electrode-donating group means a functional group that donates electron density into a bond or delocalized resonance system, and includes: O ⁇ , S ⁇ , NR 2 , NHR, NH 2 , —NHC(O)R, —OR, OH, —OC(O)R, —SR, SH, Br, I, Cl, F, R, and C 5 -C 14 aryl, where R is C 1 -C 12 alkyl.
• Electrode-withdrawing group means a functional group that removes electron density into a bond or delocalized resonance system, and includes: NO 2 , CN, CO 2 H, CO 2 R, C(O)NH 2 , C(O)NHR, C(O)NR 2 , CHO, C(O)R, SO 2 R, SO 2 OR, NO, and C 5 -C 14 aryl, where R is C 1 -C 12 alkyl (J. March (1985) Advanced Organic Chemistry, Third Ed., John Wiley & Sons, New York, p. 238).
• Heterocycle refers to a molecule with a ring system in which one or more ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur (as opposed to carbon).
• Enzymatically extendable refers to a nucleotide which is: (i) capable of being enzymatically incorporated onto the terminus of a polynucleotide chain through the action of a polymerase enzyme, and (ii) capable of supporting further primer extension.
• Enzymatically extendable nucleotides include nucleotide 5′-triphosphates, i.e. dNTP and NTP.
• Enzymatically incorporatable refers to a nucleotide which is capable of being enzymatically incorporated onto the terminus of a polynucleotide chain through the action of a polymerase enzyme.
• Enzymatically incorporatable nucleotides include dNTP, NTP, and 2′,3′-dideoxy, nucleotide 5′-triphosphates, i.e. ddNTP.
• Target sequence means a polynucleotide sequence that is the subject of hybridization with a complementary polynucleotide, e.g. a primer or probe.
• the target sequence can be composed of DNA, RNA, an analog thereof, and including combinations thereof.
• probe means a polynucleotide that is capable of forming a duplex structure by complementary base pairing with a sequence of a target nucleic acid.
• probes may be labelled, e.g. with a quencher moiety, or an energy transfer pair comprised of a fluorescent reporter and quencher.
• label refers to any moiety which can be attached to a molecule and: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g. FRET; (iii) stabilizes hybridization, i.e. duplex formation; or (iv) provides a capture moiety, i.e. affinity, antibody/antigen, ionic complexation. Labelling can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods.
• Labels include light-emitting or light-absorbing compounds which generate or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal (Kricka, L. in Nonisotopic DNA Probe Techniques (1992), Academic Press, San Diego, pp. 3-28).
• Fluorescent reporter dyes useful for labelling biomolecules include fluoresceins (U.S. Pat. Nos. 5,188,934; 6,008,379; 6,020,481), rhodamines (U.S. Pat. Nos. 5,366,860; 5,847,162; 5,936,087; 6,051,719; 6,191,278), benzophenoxazines (U.S. Pat. No.
• fluorescein dyes include 6-carboxyfluorescein; 2′,4′,1,4,-tetrachlorofluorescein; and 2′,4′,5′,7′,1,4-hexachlorofluorescein. See Example 50 and Menchen, U.S. Pat. No. 5,118,934.
• Another class of labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g. intercalators, minor-groove binders, and cross-linking functional groups (Blackburn, G. and Gait, M. Eds. “DNA and RNA structure” in Nucleic Acids in Chemistry and Biology, 2 nd Edition, (1996) Oxford University Press, pp. 15-81).
• Yet another class of labels effect the separation or immobilization of a molecule by specific or non-specific capture, for example biotin, digoxigenin, and other haptens (Andrus, A.
• energy transfer refers to the process by which the excited state energy of an excited group, e.g. fluorescent reporter dye, is conveyed through space or through bonds to another group, e.g. a quencher moiety, which may attenuate (quench) or otherwise dissipate or transfer the energy.
• Energy transfer can occur through fluorescence resonance energy transfer, direct energy transfer, and other mechanisms. The exact energy transfer mechanisms is not limiting to the present invention. It is to be understood that any reference to energy transfer in the instant application encompasses all of these mechanistically-distinct phenomena.
• “Energy transfer pair” refers to any two moieties that participate in energy transfer. Typically, one of the moieties acts as a fluorescent reporter, i.e. donor, and the other acts as a fluorescence quencher, i.e. acceptor (“Fluorescence resonance energy transfer.” Selvin P. (1995) Methods Enzymol 246:300-334; dos Remedios C. G. (1995) J. Struct. Biol. 115:175-185; “Resonance energy transfer: methods and applications.” Wu P. and Brand L. (1994) Anal Biochem 218:1-13).
• Fluorescence resonance energy transfer is a distance-dependent interaction between two moieties in which excitation energy, i.e. light, is transferred from a donor (“reporter”) to an acceptor without emission of a photon.
• the acceptor may be fluorescent and emit the transferred energy at a longer wavelength, or it may be non-fluorescent and serve to diminish the detectable fluorescence of the reporter (quenching).
• FRET may be either an intermolecular or intramolecular event, and is dependent on the inverse sixth power of the separation of the donor and acceptor, making it useful over distances comparable with the dimensions of biological macromolecules.
• the spectral properties of the energy transfer pair as a whole change in some measurable way if the distance between the moieties is altered by some critical amount.
• Self-quenching probes incorporating fluorescent donor-nonfluorescent acceptor combinations have been developed primarily for detection of proteolysis (Matayoshi, (1990) Science 247:954-958) and nucleic acid hybridization (“Detection of Energy Transfer and Fluorescence Quenching” Morrison, L., in Nonisotopic DNA Probe Techniques, L. Kricka, Ed., Academic Press, San Diego, (1992) pp. 311-352; Tyagi S. (1998) Nat. Biotechnol. 16:49-53; Tyagi S. (1996) Nat. Biotechnol 14:303-308).
• the donor and acceptor dyes are different, in which case FRET can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence.
• quenching refers to a decrease in fluorescence of a fluorescent reporter ran moiety caused by a quencher moiety by energy transfer, regardless of the mechanism. Hence, illumination of the fluorescent reporter in the presence of the quencher leads to an emission signal that is less intense than expected, or even completely absent.
• Chromatin refers to a polynucleotide including one or more modification or analog to a sugar, a nucleobase, or an internucleotide linkage.
• annealing and “hybridization” are used interchangeably and mean the base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex or other higher-ordered structure.
• the primary interaction is base specific, i.e. A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.
• solid support refers to any solid phase material upon which a nucleic acid or polypeptide is synthesized, attached or immobilized. Solid support encompasses terms such as “resin”, “solid phase”, and “support”.
• a solid support may be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof.
• a solid support may also be inorganic, such as glass, silica, controlled-pore-glass (CPG), or reverse-phase silica.
• CPG controlled-pore-glass
• the configuration of a solid support may be in the form of beads, spheres, particles, granules, or a surface.
• Solid supports may be porous or non-porous, and may have swelling or non-swelling characteristics.
• a solid support may be configured in the form of a well, depression or other vessel.
• a plurality of solid supports may be configured in an array, addressable for robotic delivery of reagents, or by detection means including scanning by laser illumination and confocal or deflective light gathering.
• Array means a predetermined spatial arrangement of polynucleotides present on a solid support or in an arrangement of vessels.
• end-point analysis refers to a method where data collection occurs only when a reaction is substantially complete.
• the term “real-time analysis” refers to periodic monitoring during PCR. Certain systems such as the ABI 7700 and 7900HT Sequence Detection Systems (Applied Biosystems, Foster City, Calif.) conduct monitoring during each thermal cycle at a pre-determined or user-defined point. Real-time analysis of PCR with FRET probes measures fluorescent dye signal changes from cycle-to-cycle, preferably minus any internal control signals.
• Novel compositions including quencher moieties are disclosed which are used to conduct, or are used to prepare other novel compositions which conduct, fluorescence-based biomolecular assays and methods.
• quencher moieties of the invention undergo efficient energy transfer with fluorescent dyes.
• the fluorescent dyes may be: (1) attached to the same biomolecule (intramolecular), or (2) attached to another biomolecule or reagent (intermolecular), which is free in solution or bound to a solid support.
• a quencher moiety can be matched with one or more fluorescent reporters to form an energy transfer pair, based on spectral overlap and other spectral properties. Typically, the fluorescent reporter in an energy transfer pair will have a longer wavelength absorbance or excitation maximum than the absorbance maximum of the quencher moiety.
• the quencher moieties of the invention include bis-diazo, triaryl structure I:
• Various counterions may be associated with charged structures I and II.
• Ar in structure II is a C 5 -C 14 aryl group, such as phenyl or substituted phenyl.
• One of the aryl carbons in each of I and II is the site of a covalent attachment, e.g. to the linker, L 1 of structure III.
• Structures I and II have extended aromaticity for efficient spectral overlap with longer wavelength (redder) fluorescent dye reporters.
• Quencher moieties of the invention have broad absorbance above 500 nm and enable energy transfer of fluorescence emitted from fluorescent dyes in the range of about 500-700 nm.
• Quenchers of the invention also have the surprising and beneficial properties of non-fluorescence and accept energy from fluorescent reporter labels by an energy-transfer mechanism.
• Quencher moieties of the invention are effective at quenching reporter dyes across a broad spectra of fluorescence detection, especially at longer wavelengths, and eliminate problems of background fluorescence resulting from direct (i.e. nonsensitized) acceptor excitation (Lee, U.S. Pat. No.
• Quencher moieties of the invention also have the surprising and unexpected properties of: ease and efficiency of preparation and purification, good chemical stability, good water-solubility, and efficient labelling of biomolecules.
• the quencher moieties of the present invention are substituted with electron-withdrawing and electron-donating substituents which induce polarity in the delocalized aryl/diazo ring systems.
• aryl carbons of structures I and II were substituted with combinations of electron-withdrawing and electron-donating groups, efficient quenching of fluorescent dyes was attained.
• At least one aryl carbon of structures I and II is substituted with an electron-withdrawing group, and at least one aryl carbon of structures I and II is substituted with an electron-donating group.
• the electron-withdrawing group may be on the same or different aryl ring as the electron-donating group.
• Electron-withdrawing groups are selected from NO 2 , CN, CF 3 , CO 2 H, CO 2 R, CONH 2 , CONHR, CONR 2 , CHO, C(O)R, SO 2 R, SO 2 OR, SO 2 CF 3 , SO 3 H, NO, and C 5 -C 14 aryl, where R is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
• Electron-donating groups are selected from O ⁇ , S ⁇ , NR 2 , NHR, NH 2 , NHCOR, OR, OH, OCOR, SR, SH, Br, I, Cl, F, C 1 -C 12 alkyl, and C 5 -C 14 aryl, where R is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
• the electron-withdrawing and electron-donating groups on the aryl carbons of the aromatic rings of I and II maybe in any configuration, i.e. ortho, meta, or para to the diazo group.
• Compounds of the present invention may be prepared by reacting aryl compounds substituted with electron-donating groups and aryl diazonium compounds.
• the diazonium compounds may be purchased from commercial sources or prepared by diazotization of aryl amines with diazotizing reagents such as NaNO 2 /HCl, nitrosylsulfuric acid, and nitrosonium tetrafluoroborate.
• the diazonium compounds can be isolated or used in situ by the subsequent addition of the aryl compound substituted with one or more electron-donating group such as an alkylamine or alkoxy group, e.g. methoxy.
• the compounds described in the Examples (1-88) may be prepared by other known reactions, using routes known in the art, or from other commercial sources. For a general review of azo dye chemistry, see: Zollinger, H. Color Chemistry; VCH, New York, 1987.
• a fluorescence quencher composition of the invention may have structure III:
• Y may be N or CR, where R is H, C 1 -C 6 alkyl or C 5 -C 14 aryl.
• the linkers, L 1 , L 2 , and L 3 are independently selected from a bond, C 1 -C 12 alkyldiyl, C 1 -C 12 alkoxyldiyl, C 1 -C 12 alkylaminodiyl, C 1 -C 12 alkylamidediyl, C 5 -C 14 aryldiyl, and 1-20 ethyleneoxy units.
• X is an amino acid, a polypeptide, a nucleoside, a nucleotide, a polynucleotide, and protected forms thereof, or an acid-labile protecting group, e.g. 4,4′-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT), trityl, substituted trityl, 9-phenylxanthen-9-yl (pixyl), and trialkylsilyl (Beaucage (1992) Tetrahedron 48:2223-2311 at 2233-2242).
• DMT 4,4′-dimethoxytrityl
• MMT 4-monomethoxytrityl
• trityl substituted trityl
• 9-phenylxanthen-9-yl pixyl
• trialkylsilyl Beaucage (1992) Tetrahedron 48:2223-2311 at 2233-2242).
• Z is selected from H, CO 2 H, OH, NH 2 , NUR, NR 2 , SH, an ester, a cleavable linker, a solid support, a reactive linking group, and a label selected from a fluorescent dye, a hybridization-stabilizing moiety, a chemiluminescent dye, and an affinity ligand.
• Q is selected from the diazo structures I and II.
• Z may be any ester, such as an oxalate, phenoxymethyl, quinone, diglycolate, succinate, or allyloxycarbonyl. Z may be selected from the structures:
• fluorescence quencher compositions include the structures:
• L 1 is alkylamidediyl
• L 2 is alkoxydiyl
• L 3 is alkyldiyl
• n is 1 to 12.
• Fluorescence quencher compositions of the invention may include a quencher moiety linked to a solid support through a cleavable linker, represented by the general structures IV and V:
• Q, Y, L 1 , L 2 , and L 3 are selected from structures I, II, and III.
• A may be any cleavable linker, including the structures:
• R′ is H, C 1 -C 12 alkyl or C 1 -C 12 alkoxy.
• the cleavable linker may be an allyl or propargyl linker, cleavable by a metal reagent, such as a palladium complex.
• L 4 and L 5 may be a bond, C 1 -C 12 alkyldiyl, C 1 -C 12 alkoxyldiyl, C 1 -C 12 alkylaminodiyl, C 1 -C 12 alkylamidediyl, C 5 -C 14 aryldiyl, or 1-20 ethyleneoxy units.
• the ester linkers A may be cleaved by basic reagents such as aqueous or gaseous ammonium hydroxide, anhydrous amines, aqueous hydroxide reagents, and aqueous amines.
• the ester linkers may be selected on the basis of their cleavage rate and desired stability of the linkage between the quencher moiety and the solid support. For example, an oxalate linkage is relatively labile, being virtually completely cleaved within a few minutes in concentrated ammonium hydroxide at room temperature.
• the succinate linkage may require one hour or more under the same conditions.
• the quinone and diglycolate linkages have intermediate stability to basic cleavage.
• Alkoxysilyl linkers may be cleaved by strong base or fluoride reagents.
• Disulfide linkers may be cleaved by reducing agents such as dithiothreitol (DTT).
• DTT dithi
• [0077] may be polystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, or copolymers and grafts of such.
• the solid support may be in any form, e.g. a particle, a bead, a membrane, a frit, a fiber, a tube, a capillary, a slide, a plate, a micromachined chip, an alkanethiol-gold layer, a magnetic bead, a non-porous surface, an addressable array, or any polynucleotide-immobilizing medium.
• G is a hybridization-stabilizing moiety, such as a minor groove binder, intercalator, polycation, such as polylysine and spermine, and cross-linking functional group.
• Hybridization-stabilizers may increase the stability of base-pairing, i.e. affinity, or the rate of hybridization (Corey (1995) J. Amer. Chem. Soc. 117:9373-74) of the primer and target, or probe and target.
• Hybridization-stabilizers serve to increase the specificity of base-pairing, exemplified by large differences in Tm between perfectly complementary oligonucleotide and target sequences and where the resulting duplex contains one or more mismatches of Watson/Crick base-pairing (Blackburn, G. and Gait, M. Eds. “DNA and RNA structure” in Nucleic Acids in Chemistry and Biology, 2 nd Edition, (1996) Oxford University Press, pp. 15-81 and 337-46).
• Minor groove binders include Hoechst 33258 (Rajur (1997) J. Org. Chem. 62:523-29), distamycin, netropsin, (Gong (1997) Biochem. and Biophys. Res. Comm. 240:557-60), and CDPI 1-3 (U.S. Pat. No. 5,801,155; WO 96/32496).
• An example of a minor groove binder is CDPI 3 , represented by the structure:
• L are the sites of attachment to L 3 and L 5 (Dempcy, WO 01/31063).
• Fluorescence quencher compositions of the invention include quencher-phosphoramidites according to the structure VI:
• Quencher-phosphoramidite reagents VI are particularly useful for the automated synthesis of labelled polynucleotides.
• R 1 and R 2 are C 1 -C 12 alkyl such as methyl, ethyl, or isopropyl; C 5 -C 14 aryl; or cycloalkyl containing up to 10 carbon atoms such as, morpholino.
• R 1 and R 2 may be C 4 -C 11 cycloalkyl, e.g. morpholino.
• R 3 is a phosphite ester protecting group which prevents unwanted extension of the polynucleotide.
• R 3 is stable to polynucleotide or polypeptide synthesis conditions yet is able to be removed from a synthetic polynucleotide product with a reagent that does not adversely affect the integrity of the polynucleotide or the dye.
• R 3 may be C 1 -C 6 alkyl, such as methyl, tert-butyl, or cyanoethyl; C 5 -C 14 aryl, such as phenyl or 2-(4-nitrophenyl)ethyl.
• Exemplary quencher-phosphoramidite 75 (Example 42) was prepared by coupling linker compound 76 with quencher NHS ester 36 by amide bond formation (Example 34) to form 77 which was phosphitylated to give 75.
• Fluorescence quencher compositions of the invention include quencher-supports where a nucleotide is attached at its 3′ or 5′ position, according to the structures VII and VIII:
• Q, Y, L 1 , L 2 , L 3 , and L 4 are selected from structures I, II, III, and IV.
• X is H or an acid-labile protecting group.
• B may be any protected or unprotected nucleobase.
• the exocyclic amino groups of nucleobase may be protected with acyl groups, e.g. isobutyryl or benzoyl, and as formamidines and acetamidines.
• R 3 may be unprotected, e.g. phosphodiester; or protected, e.g. cyanoethyl, C 1 -C 6 alkyl, or C 5 -C 14 aryl.
• Compositions VII and VIII may be intermediates formed during automated oligonucleotide synthesis on a solid support.
• Composition VII may be formed by: (1) deprotecting quenchers-support IV, i.e. removing the acid-labile protecting group X with acid, (2) coupling a 3′-phosphoramidite nucleoside, and (3) oxidizing the internucleotide phosphite linkage to phosphate.
• deprotecting quenchers-support IV i.e. removing the acid-labile protecting group X with acid
• X is DMT
• IV is treated with trichloroacetic acid or dichloroacetic acid.
• a capping step may be conducted to prevent any unreacted sites from subsequent propagation, e.g. acetylation of 5′ OH with acetic anhydride.
• Cleavage of linker A results in a 3′-quencher labelled oligonucleotide.
• Composition VIII may be formed by the same steps (1), (2), and (3), with iteration to give an oligonucleotide synthesized in the 5′ to 3′ direction, when 5′-phosphoramidites are employed in step (2). Cleavage of linker A then results in a 5′-quencher labelled oligonucleotide.
• nucleobase labelling sites include the 8-C of a purine nucleobase, the 7-C or 8-C of a 7-deazapurine nucleobase, and the 5-position of a pyrimidine nucleobase (Nardone, U.S. Pat. No. 6,117,986).
• the labelled nucleotide may be enzymatically incorporatable and enzymatically extendable by polymerases or reverse transcriptase.
• Quencher labelled nucleosides and nucleotides may have the following formula:
• quencher labelled nucleosides and nucleotides may have the following formula:
• E is an energy transfer pair comprising a reporter dye and a quencher moiety attached by a linker.
• B is a nucleobase, e.g. uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine.
• R 19 is triphosphate, thiophosphate, or phosphate ester analog.
• the quencher labelled nucleosides may be intermediates useful for conversion to nucleotide 5′-triphosphates or other compounds, where R 19 is H or an acid-labile protecting group.
• the acid-labile protecting group may be a trityl group, such as DMT.
• R 20 and R 21 when taken alone, are each independently H, HO, and F.
• Linker L may include alkynyl, propargyl, propargylethoxyamido, vinyl, and allyl groups.
• L may be:
• n 0, 1, or 2 (Khan, U.S. Pat. Nos. 5,770,716 and 5,821,356; Hobbs, U.S. Pat. No. 5,151,507).
• Polynucleotides are commonly synthesized on solid supports by the phosphoramidite method (Caruthers, U.S. Pat. No. 4,973,679; Beaucage (1992) Tetrahedron 48:2223-2311), using commercially available phosphoramidite nucleosides (Caruthers, U.S. Pat. No. 4,415,732), supports, e.g. silica, controlled-pore-glass (Caruthers, U.S. Pat. No. 4,458,066) and polystyrene (Andrus, U.S. Pat. Nos. 5,047,524 and 5,262,530) and automated synthesizers such as Models 392, 394, 3948, 3900 DNA/RNA Synthesizers (Applied Biosystems, Foster City, Calif.).
• Labelled polynucleotides may be formed by coupling a reactive linking group on a label, e.g. a quencher moiety, with a polynucleotide in a suitable solvent in which both are soluble or appreciably soluble, using methods well-known in the art.
• a reactive linking group on a label e.g. a quencher moiety
• a polynucleotide in a suitable solvent in which both are soluble or appreciably soluble
• a suitable solvent in which both are soluble or appreciably soluble
• the label may bear a reactive linking group at one of the substituent positions, e.g. an aryl-carboxyl group of a quencher moiety I or II, or the 5- or 6-carboxyl of fluorescein or rhodamine, for covalent attachment through a linkage.
• a reactive linking group at one of the substituent positions, e.g. an aryl-carboxyl group of a quencher moiety I or II, or the 5- or 6-carboxyl of fluorescein or rhodamine, for covalent attachment through a linkage.
• the linkage linking a label and the polynucleotide or nucleotide should not (i) interfere with hybridization affinity or specificity, (ii) diminish quenching, (iii) interfere with primer extension, (iv) inhibit polymerase activity, or (v) adversely affect the fluorescence or quenching properties of the label.
• Electrophilic reactive linking groups form a covalent bond with nucleophilic groups such as amines and thiols on a polynucleotide or polypeptide.
• electrophilic reactive linking groups include active esters, isothiocyanate, sulfonyl chloride, sulfonate ester, silyl halide, 2,6-dichlorotriazinyl, phosphoramidite, maleimide, haloacetyl, epoxide, alkylhalide, allyl halide, aldehyde, ketone, acylazide, anhydride, and iodoacetamide.
• Active esters include succinimidyl (NHS), hydroxybenzotriazolyl (HOBt) and pentafluorophenyl esters.
• An NHS ester of a quencher moiety may be preformed, isolated, purified, and/or characterized, or it may be formed in situ and reacted with a nucleophilic group of a polynucleotide or polypeptide.
• a quencher moiety carboxyl group is activated by reacting with some combination of: (1) a carbodiimide reagent, e.g. dicyclohexylcarbodiimide, diisopropylcarbodiimide, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide); or a uronium reagent, e.g.
• TSTU (O-(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate
• HBTU (O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate)
• HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
• an activator such as HOBt (1-hydroxybenzotriazole) or HOAt (1-hydroxy-7-azabenzotriazole
• N-hydroxysuccinimide to give the NHS ester.
• the general quencher-phosphoramidite VI reacts with a hydroxyl group, e.g. 5′ terminal OH of a polynucleotide bound to a solid support, under mild acid activation, to form an internucleotide phosphite group which is then oxidized to an internucleotide phosphate group.
• the quencher-phosphoramidite reagent reacts with a hydroxyl group on the side chain of an amino acid of a polypeptide such as serine, threonine, or tyrosine.
• the quencher moiety contains functional groups that require protection either during the synthesis of the phosphoramidite reagent or during its subsequent use to label polynucleotides or polypeptides.
• the protecting group(s) used will depend upon the nature of the functional groups, and will be apparent to those having skill in the art (Greene, T. and Wuts, P. Protective Groups in Organic Synthesis, 2nd Ed., John Wiley & Sons, New York, 1991).
• the quencher will be attached at the 5′ terminus of the polynucleotide, as a consequence of the common 3′ to 5′ direction of synthesis method.
• the 3′ terminus of a polynucleotide may be labelled with a quencher-phosphoramidite when synthesis is conducted in the less-common 5′ to 3′ direction with 3′-protected, 5′ phosphoramidite nucleosides (Wagner, (1997) Nucleosides & Nucleotides 16:1657-60).
• Other quencher-phosphoramidite reagents, nucleosidic and non-nucleosidic allow for labelling at other sites of a polynucleotide, e.g. 3′ terminus, nucleobase, internucleotide linkage, sugar. Labelling at the nucleobase, internucleotide linkage, and sugar sites allows for internal and multiple labelling.
• a nucleobase-labelled polynucleotide may have the following formula:
• nucleobase-labelled polynucleotide may have the following formula:
• E is an energy transfer pair comprising a reporter dye and a quencher moiety attached by a linker.
• B is a nucleobase, e.g. uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine.
• L is a linker, e.g. propargyl, propargylethoxyamido, allyl, vinyl, or C 1 -C 12 alkyldiyl.
• R 21 is H, OH, halide, azide, amine, C 1 -C 6 aminoalkyl, C 1 -C 6 alkyl, allyl, C 1 -C 6 alkoxy, —OCH 3 , or —OCH 2 CH ⁇ CH 2 .
• R 22 is H, phosphate, internucleotide phosphodiester, or internucleotide analog.
• R 23 is H, phosphate, internucleotide phosphodiester, or internucleotide analog.
• the nucleobase-labelled polynucleotide may bear multiple fluorescent labels, e.g. dyes, attached through the nucleobases.
• Nucleobase-labelled polynucleotides may be formed by: (i) enzymatic incorporation of enzymatically incorporatable nucleotide reagents where R 19 is triphosphate, by a DNA polymerase or ligase, and (ii) coupling of a nucleoside phosphoramidite reagent by automated synthesis (Theisen (1992) “Fluorescent dye phosphoramidite labelling of polynucleotides”, in Nucleic Acid Symposium Series No. 27, Oxford University Press, Oxford, pp. 99-100).
• 5′ quencher-labelled polynucleotides of the invention include the following structure:
• Q is structure I or II.
• X is O, NH, or S;
• R 21 is H, OH, halide, azide, amine, C 1 -C 6 aminoalkyl, C 1 -C 6 alkyl, allyl, C 1 -C 6 alkoxy, —OCH 3 , or —OCH 2 CH ⁇ CH 2 ;
• R 22 is internucleotide phosphodiester, or internucleotide analog.
• L is a linker, including C 1 -C 12 alkyldiyl, e.g. n-hexyldiyl, aryldiyl, or polyethyleneoxy (U.S. Pat. No.
• the 5′ quencher-polynucleotide may have additional covalently attached labels, such as a fluorescent dye or a minor groove binder.
• a solid support bearing a quencher of the present invention can be utilized as a solid support for polynucleotide synthesis (U.S. Pat. Nos. 5,141,813; 5,231,191; 5,401,837; 5,736,626).
• the quencher or the functionality is present during synthesis of the polynucleotide.
• the quencher or the functionality remains covalently attached to the polynucleotide.
• Polynucleotides labelled at the 3′ terminus may have the following structure:
• the probe comprises 2 to 100 nucleotides.
• Q is structure I or II.
• B is any nucleobase, e.g. uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine.
• L is a linker, including C 1 -C 12 alkyldiyl, e.g. n-hexyldiyl, aryldiyl, or polyethyleneoxy.
• R 21 is H, OH, halide, azide, amine, C 1 -C 6 aminoalkyl, C 1 -C 6 alkyl, allyl, C 1 -C 6 alkoxy, —OCH 3 , or —OCH 2 CH ⁇ CH 2 .
• R 23 is internucleotide phosphodiester or internucleotide analog.
• the 3′ quencher-polynucleotide may have additional covalently attached labels, such as a fluorescent dye or a minor groove binder.
• 3′ quencher-polynucleotides bound to a solid support may be derived from VII and have the general structure:
• polynucleotide comprises 2 to 100 nucleotides.
• 5′ quencher labelled polynucleotides bound to a solid support may be derived from VIII and have the general structure:
• polynucleotide comprises 2 to 100 nucleotides.
• Polynucleotides can be immobilized on solid supports by a covalent bond attachment, ionic interaction, hybridization to an immobilized probe, hydrophobic interaction, or ligand/receptor interaction, using a variety of known procedures (Lane, U.S. Pat. No. 5,902,724; Mirkin, WO 98/04740). Covalent bonds between a polynucleotide and solid support include Schiff-base type linkages (Lukhtanov, WO 01/09385) and phosphoramidate linkages (Rampal, U.S. Pat. No. 6,013,789).
• Nucleic acid analogs are structural analogs of DNA and RNA and which are designed to hybridize to complementary nucleic acid sequences. Through modification of the internucleotide linkage, the sugar, and/or the nucleobase, nucleic acid analogs of the invention may attain any or all of the following desired properties: 1) optimized hybridization specificity or affinity, 2) nuclease resistance, 3) chemical stability, 4) solubility, 5) membrane-permeability, and 6) ease or low costs of synthesis and purification.
• PNA peptide nucleic acids
• the N-[2-(aminoethyl)]glycine polyamide linkage with nucleobases attached to nitrogen through a methylene carbonyl linkage by an amide bond has been well-studied as an embodiment of PNA and shown to possess exceptional hybridization specificity and affinity (Buchardt, WO 92/20702; Nielsen (1991) Science 254:1497-1500; Egholm (1993) Nature, 365:566-68; Nielsen, P. and Egholm, M. (1999) in Peptide Nucleic Acids; Protocols and Applications, Horizon Scientific Press, Norfolk, England).
• PNA may be synthesized at any scale, e.g. 2-100 ⁇ mole scale or more, using Fmoc/Bhoc, tBoc/Z, or MMT protecting group monomers on an Expedite Synthesizer (Applied Biosystems) on XAL or PAL support; or on the Model 433A Synthesizer (Applied Biosystems) with MBHA support; or on other automated synthesizers.
• PNA may be synthesized on many of the solid supports commonly used for peptide synthesis (M. W. Pennington and B. M. Dunn (Eds.) “Methods in molecular biology, Vol. 35: Peptide synthesis protocols”, Humana Press, Totowa, N.J.
• PNA-DNA chimera are oligomer molecules with discrete PNA and nucleotide moieties. They can be synthesized by covalently linking PNA monomers and nucleotides in virtually any combination or sequence. Efficient and automated methods have been developed for synthesizing PNA-DNA chimera (Vinayak (1997) Nucleosides & Nucleotides 16:1653-56; Uhlmann (1996) Angew. Chem., Intl. Ed. Eng. 35:2632-35; Uhlmann, EP 829542; Van der Laan (1997) Tetrahedron Lett. 38:2249-52; Van der Laan (1998) Bioorg. Med. Chem. Lett. 8:663-68.
• PNA-DNA chimera are designed to have desirable properties found in PNA and DNA, e.g. superior hybridization properties of PNA and biological functions like DNA, including primer extension through the 3′ OH terminus of the DNA moiety (Uhlmann (1998) Biol. Chem. 379:1045-52).
• PNA and PNA-DNA chimera can be labelled with a quencher moiety I or II, and a fluorescent reporter, to form PNA FRET probes (Hyldig-Nielsen, U.S. Pat. No. 5,985,563; Coull, WO 98/24933; Coull, WO 99/22018; Gildea, WO 99/21881; Coull, WO 99/49293).
• the linker between a PNA monomer unit and a label is: (i) a covalent bond; (ii) an C 1 -C 12 alkyldiyl; (iii) ethyleneoxy —(CH 2 CH 2 O) n —, where n is 1 to 12, (iv) C 5 -C 14 aryldiyl; (v) 2-(2-aminoethoxy)acetic acid; or (vi) one or more amino acids (Gildea (1998) Tetrahedron Letters 39:7255-58). Lysine, aspartic acid, and glutamic acid side chains in the linker may be quencher-linkage sites in quencher-labelled PNA.
• the ⁇ -amino group of the sidechain of lysine may be the reactive linking group for attachment of a label, e.g. fluorescent reporter or quencher moiety.
• Linkers are typically attached to the amino and/or carboxyl terminus of the PNA by the corresponding monomer units with compatible protecting groups and reactive functionality for condensation with PNA monomer units and the solid support.
• the “O linker”, units of 2-(2-aminoethoxy)acetic acid can be attached to the amino terminus of any PNA backbone amino group, or on amino functionality of a solid support.
• Polypeptides that are labeled with a quencher of the invention can be used in both in vivo and in vitro enzymatic assays.
• the present invention provides generally for methods to determine if a sample contains an enzyme.
• a method may comprise: (a) contacting the sample with a polypeptide labelled with a fluorescent reporter and a quencher moiety; (b) exciting the fluorophore; and (c) determining a fluorescence property of the sample, wherein the presence of the enzyme in the sample results in a change in the fluorescence property.
• the change in fluorescence may be due to cleavage or assembly of a recognition site for the enzyme.
• the degree of donor-acceptor energy transfer in the sample can be determined, for example, as a function of the amount of fluorescence from the donor moiety, the amount of fluorescence from the acceptor moiety, the ratio of the amount of fluorescence from the donor moiety to the amount of fluorescence from the acceptor moiety or the excitation state lifetime of the donor moiety.
• the assay methods can also be used to determine whether a compound alters the activity of an enzyme, i.e. screening assays, and determining the amount of activity of an enzyme in a sample from an organism.
• the amount of enzyme activity in the sample is determined as a function of the degree of donor-acceptor energy transfer in the sample and the amount of activity in the sample is compared with a standard activity for the same or different concentration of enzyme.
• Representative enzymes with which the present invention can be practiced include, for example, trypsin, enterokinase, HTV-1 protease, prohormone convertase, kininogenase, and proteases in general.
• the most convenient assays for proteases are based on donor-acceptor energy transfer from a donor fluorophore to a quencher placed at opposite ends of a short peptide chain containing the potential cleavage site (Knight C. G., (1995) Methods in Enzymol. 248:1 8-34).
• Proteolysis separates the fluorophore and quencher, resulting in increased intensity in the emission of the donor fluorophore.
• the polypeptide sequence includes a cleavage recognition site, specific for an enzyme or other cleavage agent of interest.
• a cleavage recognition sequence for a protease is a specific amino acid sequence recognized by the protease during proteolytic cleavage.
• Many protease cleavage sites are known in the art, and these and other cleavage sites can be included in the linker moiety (Matayoshi, (1990) Science 247:954; Dunn in Meth. in Enzymol. 241:254(1994); Seidahin Meth. in Enzymol. 244: 175 (1994); Thomberryin Meth. in Enzymol.
• Cleavage of the sequence results in separation of the fluorescent dye and the quencher moiety of a FRET peptide.
• the cleavage may be measurable as a change in donor-acceptor energy transfer, with a resulting increase in detectable fluorescence.
• peptide assembly can be detected by an increase in donor-acceptor energy transfer (quenching) between a peptide fragment bearing quencher moiety and a peptide fragment bearing a fluorescent reporter.
• An electrophilic reactive linking group of a quencher moiety of the invention can react with a nucleophilic side chain of an amino acid in a polypeptide (Brinkley, 1992, Bioconjugate Chem. 3:2).
• a-amino groups are less basic than lysines and are reactive at around pH 7.0. Sometimes they can be selectively modified in the presence of lysines. There is usually at least one ⁇ -amino acid in a protein, and in the case of proteins that have multiple peptide chains or several subunits, there can be more (one for each peptide chain or subunit). Since either N-terminal amines or lysines are almost always present in any given protein or peptide, and since they are easily reacted, the most commonly used method of protein modification is through these aliphatic amine groups.
• the quencher moiety may be conjugated to the N-terminus of a resin-bound polypeptide before removal of other protecting groups and release of the labeled peptide from the resin. About five equivalents of an amine-reactive quencher reagent may be used per amine of the immobilized peptide (Haugland, 1996, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.)
• Another reactive linking group in many polypeptides is a thiol residue from the sulfur-containing amino acid cystine and its reduction product cysteine (or half-cystine), which are counted together as one of the 20 amino acids.
• Cysteine contains a free thiol group, which is more nucleophilic than amines and is generally the most reactive functional group in a protein. It reacts with some of the same modification reagents as do amines and in addition can react with reagents that are not very reactive toward amines.
• thiols are reactive at neutral pH, and therefore they can be coupled to other molecules selectively in the presence of amines.
• proteins with these groups are relatively reactive, proteins with these groups often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Reduction of the disulfide bonds with a reagent such as dithiothreitol (DTT) is required to generate the reactive free thiol.
• DTT dithiothreitol
• cystine and cysteine some proteins also have the amino acid methionine, which contains sulfur in a thioether linkage.
• Acylation reactions may be performed at polypeptide amino groups with quencher reagents. Acylation reactions may be described by the following general scheme:
• Pep is the polypeptide
• X is a leaving group
• Q is the quencher moiety.
• the reactive reagent Q—CO—X may be produced in situ by the action of an activating agent, such as a carbodiimide, on the free carboxylic acid, but more preferable is the use of stable active esters that may be stored as solid reagents, e.g. N-hydroxysuccinimide (NHS) esters, and also more water-soluble sulfonated forms.
• an activating agent such as a carbodiimide
• amine-reactive reagents which may serve as reactive linking groups for the quencher moiety to prepare quencher-labelled polypeptide include other succinimidyl esters, isothiocyanates (—NCS), sulphonyl halides (—SO 2 Cl) and dichlorotriazine derivatives.
• Thiol-reactive reagents include iodoacetyl (—C(O)CH 2 I) and maleimido derivatives.
• Oligonucleotides labelled with a fluorescent dye and a quencher of the invention are useful in a wide variety of important nucleic acid hybridization and amplification applications, including allelic discrimination and real-time PCR quantitation.
• the specificity of such oligonucleotides as primers and probes is effective for distinguishing single base-pair differences and mismatches.
• the fluorescent/quencher probes of the invention are useful as detection agents in a variety of DNA amplification/quantification strategies including, for example, 5′-nuclease assay, Strand Displacement Amplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA), Rolling Circle Amplification (RCA), Oligonucleotide Ligation Assay (OLA), Ligase Chain Reaction (LCR), Transcription-Mediated Amplification (TMA) and Q-beta replicase.
• SDA Strand Displacement Amplification
• NASBA Nucleic Acid Sequence-Based Amplification
• RCA Rolling Circle Amplification
• OOA Oligonucleotide Ligation Assay
• LCR Ligase Chain Reaction
• TMA Transcription-Mediated Amplification
• Q-beta replicase Q-beta replicase.
• Fluorescent/quencher probes are also useful for direct detection of targets in other solution phase or solid phase (e.g., array) assays.
• the fluorescent/quencher probes can be used in any format, including, for example, molecular beacons, Scorpion probesTM, Sunrise probesTM, light up probes, InvaderTM Detection probes, and TaqManTM probes. See, for example, Cardullo, R. (1988) Proc. Natl. Acad. Sci. USA, 85:8790-8794; Stryer, L., (1978) Ann. Rev. Biochem., 47:819-846; Rehman, F. N., (1999) Nucleic Acids Research, 27:649-655; Gibson, E. M., (1996) Genome Methods, 6:995-1001; Livak, U.S. Pat. No.
• a method of the invention includes nucleic acid amplification, i.e. amplifying a target polynucleotide, i.e. template DNA, with nucleotide 5′-triphosphates, a polymerase, and two or more primers.
• the primers are complementary to the target polynucleotide sequence.
• One or more of the primers can be covalently attached by a linkage to an aryl carbon of a quencher moiety I or II.
• a detectable probe, complementary to the target polynucleotide sequence may also be included in the amplification reaction.
• the process uses the 5′ to 3′ exonuclease activity of TAQ polymerase, and other thermo-stable polymerases, to digest the double-labelled, self-quenched, detectable probe during the PCR amplification process.
• the probe is cleaved by the 5′-exonuclease activity of DNA polymerase if, and only if, it hybridizes within the target sequence being amplified, i.e. between the primer-binding sites. Cleavage of the probe generates an increase in the fluorescence intensity of the fluorescent reporter.
• the fluorescent donor and quencher are preferably each located at or near the 3′- and 5′-ends of the probe.
• a probe may be designed with a 5′ fluorescent reporter dye, e.g. carboxyfluorescein, and a 3′ quencher moiety of the present invention.
• the Tm of the probe (about 65° C.) is selected primarily on the basis of length and G+C content and known algorithms to insure binding to the target polynucleotide under extension conditions.
• the detectable probe is synthesized using a 3′-quencher support, e.g. 65 (Example 46) or 69 (Example 47).
• the dual-labelled, detectable probe is synthesized on the support including the step of covalent attachment of the fluorescent reporter.
• the fluorescent reporter can be attached via a reactive linking group, e.g.
• the 3′-terminal nucleotide of the probe is blocked or rendered incapable of extension by a nucleic acid polymerase. Such blocking is achieved conveniently by the quencher moiety or the fluorescent reporter at the terminal 3′-hydroxyl. Alternatively, the 3′ hydroxyl may be phosphorylated or blocked by other functionality.
• One method of the invention includes nucleic acid amplification, i.e. amplifying a target polynucleotide with nucleotide 5′-triphosphates, a polymerase, two or more primers, and a detectable probe.
• the detectable probe may be labelled with a fluorescent reporter dye and a quencher moiety.
• the primers are complementary to the target polynucleotide sequence.
• the probe is complementary to the target polynucleotide at a binding site between the primer binding sites.
• the probe is covalently attached by a linkage to an aryl carbon of a quencher moiety I or II.
• a signal can be detected from the detectable probe, either upon hybridization of the probe to the target, or upon cleavage of the probe by the polymerase, during amplification.
• Cleavage of the dual labelled, self-quenching probe by TAQ polymerase with 5′-nuclease activity generates free fluorescent reporter each cycle of amplification, leading to exponential accumulation of fluorescent signal, correlated to the exponential production of PCR product (“amplicon”).
• amplicon correlated to the exponential production of PCR product
• CT threshold cycle
• CT is designated, as the fractional cycle number, at which the increase in fluorescence crosses some fixed threshold above baseline.
• a series of 5′-fluorescent reporter, 3′-quencher probes were synthesized with a 3′ quencher support 69 (Example 48) and fluorescent dye phosphoramidites for the 5′ labelling (Example 50). These probes were used in the 5′ nuclease assay, i.e. “TaqMan”, on the ABI Prism 7700 instrument (Example 51). By way of comparison, the same probe sequence was labelled with the same set of 5′ fluorescent dyes. The variable of study was the quencher moiety. The second set of probes was labelled with TAMRA (tetramethylrhodamine), a fluorescent quencher.
• TAMRA tetramethylrhodamine
• Probes containing a fluorescent dye-quencher pair have been developed for hybridization assays where the probe forms a hairpin structure due to a self-complementary sequence.
• a hairpin probe hybridizes to itself to form a stem/loop structure.
• the quencher molecule may brought into proximity with the fluorescent reporter dye to effectively quench the fluorescence of the fluorescent dye (WO 90/03446; EP 0601889 A2).
• hybridization of the hairpin FRET probe to the complementary target sequence disrupts the hairpin structure and causes the probe to adopt a conformation where the quencher molecule is no longer close enough to the reporter molecule to quench the reporter molecule.
• the probes provide an increased fluorescent signal when hybridized to a target sequence than when they are unhybridized (Tyagi (1996) (Nature Biotech., 14:303-309; U.S. Pat. Nos. 5,119,801 and 5,312,728).
• the hybridization probe may be labelled at the 5′ or 3′ terminus with the fluorescent dye and at the other terminus with a quencher moiety of the invention.
• the probes for hybridization detection may comprise a nucleic acid analog, e.g. one or more PNA (N-[2-(aminoethyl)]glycine) units having a nucleobase attached to nitrogen through a methylene carbonyl linkage, or LNA (2′-4′ or 3′-4′ bicyclic sugar modifications).
• PNA N-[2-(aminoethyl)]glycine
• LNA 2′-4′ or 3′-4′ bicyclic sugar modifications
• Polynucleotide fragments labelled with quencher moieties I and II may be generated through template-directed enzymatic synthesis using quencher-labelled primers or quencher-labelled nucleotides, e.g. by ligation or polymerase-directed primer extension.
• the resulting polynucleotide fragments may be further labelled with fluorescent reporters to form energy-transfer pairs.
• Quencher labelled polynucleotide fragments may serve to intermolecularly quench the fluorescence of other fluorescent-labelled compounds.
• Polynucleotide fragments may be subjected to a size-dependent separation process, e.g., electrophoresis or chromatography, and the separated fragments are detected subsequent to the separation, e.g., by laser-induced fluorescence (Hunkapiller, U.S. Pat. No. 4,811,218).
• a size-dependent separation process e.g., electrophoresis or chromatography
• Multiple classes of polynucleotides may be separated simultaneously and the different classes are distinguished by spectrally resolvable fluorescent reporters.
• electrophoresis the classes separate on the basis of electrophoretic migration rate.
• chain termination methods of DNA sequencing i.e. dideoxy DNA sequencing, or Sanger-type sequencing, and fragment analysis maybe employed (Sanger (1977) “DNA sequencing with chain-terminating inhibitors”, Proc. Natl. Acad. Sci. USA 74:5463-5467) with the fluorescence quenching compositions of the invention
• Exemplary chain-terminating nucleotide analogs “terminators” include the 2′,3′-dideoxynucleoside 5′-triphosphates (ddNTP) which lack the 3′-OH group necessary for 3′ to 5′ DNA chain elongation.
• a primer or ddNTP may be labelled with a quencher moiety and a fluorescent reporter to form an energy transfer pair within the primer extension product.
• the change in fluorescence from each fragment may be detected after separation of the fragments by high-resolution electrophoresis.
• a ligation event may be detected and monitored with oligonucleotide probes labelled with quencher moieties I and II.
• Covalent joining of nucleic acid probes by ligase enzymes is one of the most useful tools available to molecular biologists.
• a phosphodiester bond can be formed between a 5′ terminus of one probe and the 3′ terminus of the other probe by a ligase enzyme, (Whiteley, U.S. Pat. No.
• Oligonucleotide ligation assays detect the presence of specific sequences in target DNA sample. Where one probe is labelled with a fluorescent reporter and the other probe is labelled with a quencher moiety of the invention, the ligation product may be detected by fluorescence. Ligation products may be separated by electrophoresis, chromatography, or other size- or charge-based separation method.
• a quencher-oligonucleotide of the present invention may be immobilized on a solid support and used as a capture probe.
• the probe may be covalently attached directly to a solid support, for example by attachment of the 3′- or 5′-terminal nucleotide of the probe to the solid support.
• the probe may be attached to the solid support by a linker which serves to distance the probe from the solid support.
• the linker may be about 5 to about 30 atoms in length, or more.
• Examples of preferred types of solid supports for immobilization of the oligonucleotide probe include controlled pore glass, glass plates, polystyrene, avidin-coated beads, cellulose, nylon, polystyrene grafted with polyethylene glycol, polyacrylamide gel and activated dextran. These solid supports are preferred for hybridization and diagnostic studies because of their chemical stability, ease of functionalization and well defined surface area. Solid supports such as controlled pore glass (CPG) and non-swelling, high cross-linked polystyrene are advantageous due to their compatibility with oligonucleotide synthesis, precluding the necessity to cleave and reattach the oligonucleotide.
• CPG controlled pore glass
• non-swelling, high cross-linked polystyrene are advantageous due to their compatibility with oligonucleotide synthesis, precluding the necessity to cleave and reattach the oligonucleotide.
• the probe may be attached to the solid support through a variety of structures and synthetic routes.
• the probe may be attached to the solid support by attachment of the 3′ or 5′ hydroxyl of the terminal nucleotide of the probe to the solid support.
• the probe is attached to the solid support through a nucleobase, a sugar, or an internucleotide linkage.
• the solid support is used as the synthesis support in preparing the fluorescent/quencher oligonucleotide probe.
• the linker arm can be attached to the 3′-OH of the 3′-terminus by an ester linkage which can be cleaved with basic reagents to free the oligonucleotide from the solid support.
• linkers are known in the art which may be used to attach the oligonucleotide probe to the solid support.
• the linker should not significantly interfere with the hybridization of the target sequence to the probe attached to the solid support.
• the linker may be formed of a non-target complementary oligonucleotide which can be readily added on to the linker by automated synthesis.
• polymers such as functionalized polyethylene glycol can be used as the linker.
• Polyethylene glycol is commercially available, soluble in both organic and aqueous media, easy to functionalized, and stable under oligonucleotide synthesis and post-synthesis conditions.
• the linker between the solid support and the probe is preferably not cleaved during removal of base protecting groups under basic conditions at high temperature.
• These linkers can, however, be selected from groups that are cleavable under a variety of conditions.
• Exemplary linkers include carbamate and amide functionality.
• kits comprising polynucleotides labelled with quencher moieties of structures I or II, and other reagents such as nucleotide 5′-triphosphates, polymerase, primers, a detectable probe, and buffer.
• Such kits are useful in primer extension, hybridization assay, and 5′ nuclease “TaqMan®” PCR detection assay.
• the kits may farther comprise one or more target polynucleotides, or standard/control polynucleotides.
• kits are useful for coupling fluorescence quencher compositions of the invention to polynucleotides and polypeptides.
• kits generally comprise a fluorescence quencher composition with a reactive linking group, e.g. a phosphoramidite or active ester, and any activator, coupling agent, buffer, solvent, or other reagent necessary to effect coupling to a polynucleotide or polypeptide.
• a reactive linking group e.g. a phosphoramidite or active ester
• an activator, coupling agent, buffer, solvent, or other reagent necessary to effect coupling to a polynucleotide or polypeptide.
• kits may contain a fluorescence quencher on solid-support and other reagents for the synthesis of a 3′-quencher labelled polynucleotide.
• N-ethylaniline 5 (2.0 gm, 0.016 moles), triethylamine (5.0 ml, 0.036 moles), and ethyl 4-bromobutyrate (3.45 gm, 0.018 moles) were heated at 110° C. for 14 hours under argon. The mixture was cooled to room temperature, diluted with 50 ml ethyl acetate, washed with 50 ml water and 50 ml saturated NaCl, dried over MgSO 4 , filtered, and evaporated under reduced pressure to give crude ethyl 4-(ethylphenylamino)butanoate 6 (3.0 gm, 77% yield).
• N-Ethylaniline 5 (2 g, 16.52 mmol) was added to a stirred suspension of sodium hydride (436 mg, 18.17 mmol) in DMF (20 ml) at ambient temperature under argon. After 45 minutes, bromomethyl acetate (3.02 g, 18.17 mmol) was added and the reaction mixture was stirred for 22 hr. DMF was removed under reduced pressure and the residue was treated with ethyl acetate (60 ml) and water (50 ml).
• N-Ethyl-1-naphthylamine 8 (11.4 gm, 0.066 moles), diisopropylethylamine (11.2 gm, 0.087 moles), and ethyl 4-bromobutyrate (19 gm, 0.097 moles) were heated at 110-120° C. for 16 hours under argon at which time, HPLC indicated the reaction was complete. The mixture solidified upon cooling to room temperature. The solid was partitioned between DCM and water. The aqueous layer was extracted with two portions of DCM.
• 3-Methoxyaniline 12 (5 gm, 0.041 moles) was dissolved in 30 ml tetrahydrofuran (THF) at room temperature under argon. Acetic anhydride (5.8 ml, 0.061 moles) was added. After stirring for 2 hours, the mixture was evaporated under reduced pressure, dissolved in several mls of toluene and purified by flash chromatography on silica gel, eluting with a mixture of ethyl acetate and hexane. 3-Methoxyacetanilide 13 (6.63 gm, 99% yield) was isolated after removing the solvent.
• 2,5-Dimethylacetanilide 17 (6.67 gm, 0.041 moles) was dissolved with heating in 65 ml THF and stirred under argon. The solution was cooled to room temperature and borane-methylsulfide complex (52 ml, 2.0M, 0.10 moles) was added by syringe over 10 minutes. After 10 minutes at ambient temperature, the mixture was heated to reflux for 3 hours. After cooling to room temperature, 40 ml water was cautiously added with stirring over 10 minutes, followed by 3.6 ml conc. HCl. After heating for 30 minutes, 100 ml DCM and 67 ml 2N NaOH were added sequentially with vigorous stirring. An emulsion resulted which was eventually separated.
• borane-methylsulfide complex 52 ml, 2.0M, 0.10 moles
• the aqueous portion was extracted with three 50 ml portions of DCM which were combined, dried over MgSO 4 , and filtered. After concentration by rotary evaporation under reduced pressure, the crude product was purified by flash chromatography on silica gel, eluting with a gradient mixture of ethyl acetate and hexane. (2,5-Dimethylphenyl)ethylamine 18 (5.36 gm, 88% yield) was isolated after removing the solvent as a colorless oil.
• N-(2-Methoxy-5-nitrophenyl)acetamide 24 ( 1 H NMR (CDCl 3 ) ⁇ 9.20, 1H, br s; 7.99, 1H, d; 6.95, 1H, d; 4.02, 3H, s; 3.12, 1H, s; 2.26, 3H, s), which was suspended in 115 ml ethanol. 5% Palladium on carbon (8.05 gm) was added, followed by the slow addition of 8.05 ml hydrazine hydrate (1 ml/5 minutes). After 90 minutes of heating, TLC indicated the reduction was complete.
• N-(3-amino-6-methoxyphenyl)acetamide 25 (1.0 gm, 0.0055 moles) was dissolved in 30 ml DCM at room temperature under argon with stirring. Diisopropylethylamine (4.83 ml, 0.028 moles) and dimethylsulfate (2.36 ml, 0.025 moles) were added. After stirring at room temperature for 8 hours, 30 ml DCM and 40 ml 0.3 N NaOH were added. The mixture was partitioned and the aqueous layer was washed with three 30 ml portions of DCM.
• N-(3-dimethylamino-6-methoxyphenyl)acetamide 26 (0.10 gm, 0.48 mmoles) was dissolved in 2 ml THF and stirred under argon at room temperature.
• Borane-methylsulfide complex (0.6 ml, 2.0M, 1.2 mmoles) was added by syringe. After 10 minutes at ambient temperature, the mixture was heated to reflux for 3 hours. After cooling to room temperature, 0.6 ml water was cautiously added with stirring 0.1 ml conc. HCl. After heating for 1 hour, 5 ml DCM and 0.3 ml 2N NaOH were added sequentially with vigorous stirring.
• Diazo-diaryl ester 47 (480 mg, 0.98 mmole) was stirred at room temperature in 6 ml THF. Lithium hydroxide hydrate (0.164 gm, 3.9 mmoles) in 3 ml water was added. The mixture was stirred overnight, then 10 ml 1 N acetic acid/sodium acetate buffer (pH 5.0) was added. The mixture was extracted with three portions of DCM. The combined organic extract was washed with saturated NaCl, dried over Na 2 SO 4 , filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography to give diazo,diaryl acid 48 (0.324 gm, 72%) after removal of solvent. Absorbance max.
• diazo-diaryl ester 43 (420 mg, 0.91 mmole) was stirred at room temperature in 6 ml THF. Lithium hydroxide hydrate (0.153 gm, 3.6 mmoles) in 3 ml water was added. The mixture was stirred overnight, then 10 ml 1 N acetic acid/sodium acetate buffer (pH 5.0) was added. The mixture was extracted with three portions of DCM. The combined organic extract was washed with saturated NaCl, dried over Na 2 SO 4 , filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography to give diazo,diaryl acid 44 (0.346 gm, 88%) after removal of solvent. Absorbance max. 537 nm (methanol); 556 nm (0.1M TEAA).
• diazo-diaryl ester 49 (145 mg, 0.31 mmole) was stirred at room temperature in 4 ml THF. Lithium hydroxide hydrate (52 mg, 1.2 mmoles) in 1.5 ml water was added. The mixture was stirred overnight, then 10 ml 1N acetic acid/sodium acetate buffer (pH 5.0) was added. The mixture was extracted with three portions of DCM. The combined organic extract was washed with saturated NaCl, dried over Na 2 SO 4 , filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography to give diazo,diaryl acid 50 (112 mg, 82%) after removal of solvent. Abs. max. 577 nm (methanol), 584 nm (0.1M TEAA).
• Diazo,diaryl acid 50 (106 mg, 0.24 mmoles) was suspended in 2.5 ml DCM at room temperature under argon with stirring. Triethylamine (67 ⁇ l, 0.5 mmoles) and pentafluorophenyl trifluoroacetate (53 ⁇ l, 0.31 mmoles) and the mixture was stirred overnight. The mixture was concentrated under vacuum and purified by silica gel chromatography, eluting with ethyl acetate and hexane to give the bis-diazo,triaryl PFP ester 51 (117 mg, 80%) after removal of solvent.
• diazo-diaryl ester 52 (230 mg, 0.45 mmole) was stirred at room temperature in 4 ml THF. Lithium hydroxide hydrate (75 mg, 1.8 mmoles) in 1.5 ml water was added. The mixture was stirred overnight, then 10 ml 1N acetic acid/sodium acetate buffer (pH 5.0) was added. The mixture was extracted with three portions of DCM. The combined organic extract was washed with saturated NaCl, dried over Na 2 SO 4 , filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography to give diazo,diaryl acid 53 (221 mg, 100%) after removal of solvent. Abs. max. 550 nm (methanol), 556 nm (0.1M TEAA).
• diazo-diaryl ester 54 (385 mg, 0.80 mmole) was stirred at room temperature in 6 ml THF. Lithium hydroxide hydrate (134 mg, 3.2 mmoles) in 3 ml water was added. The mixture was stirred overnight, then 10 ml 1 N acetic acid/sodium acetate buffer (pH 5.0) was added. The mixture was extracted with three portions of DCM. The combined organic extract was washed with saturated NaCl, dried over Na 2 SO 4 , filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography to give diazo,diaryl acid 55 (197 mg, 55%) after removal of solvent. Abs. max. 543 nm (methanol), 554 nm (0.1M TEAA).
• diazo-diaryl 61 (650 mg, 2.35 mmoles) was diazotized and reacted with ethyl 4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (632 mg, 2.14 mmoles) to give diazo-diaryl compound 63 (0.34 gm, 27%).
• diazo-diaryl 72 (768 mg, 2.35 mmoles) was diazotized and reacted with ethyl 4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (632 mg, 2.14 mmoles) to give diazo-diaryl compound 64 (0.17 gm, 12%). Absorbance max. 582 nm (methanol).
• Tetramethoxy bis-diazo,triaryl compound 30 is prepared from Fast Black K salt 29 and ethyl 4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (Example 1) by the procedure of Example 31.
• the aqueous layer was extracted with two 20 ml portions of DCM.
• the combined organic extracts were combined, dried over MgSO 4 , filtered, and concentrated under reduced pressure.
• the residue was purified by silica gel chromatography, eluting with a gradient of ethyl acetate and hexane, then methanol and DCM to give bis-diazo,triaryl 70 (950 mg, 76%) after removal of solvent as a blue-purple solid.
• Hydroxyl-quencher compound 77 (1.97 gm, 1.67 mmoles) and diisopropylammonium 5-H-tetrazolide (0.337 gm, 1.69 mmoles) are dissolved in 55 ml DCM.
• 2-Cyanoethyl-N,N-tetraisopropyl phosphoramidite ((iPr 2 N) 2 —P—OCH 2 CH 2 CN, 1.28 ml, 1.206 gm, 4.00 mmoles) was added.
• Fmoc linker compound 82 (3 g, 4.46 mmol) was treated with tert-butyl dimethylsilyl chloride (807 mg, 5.35 mmol) and imidazole (425 mg, 6.24 mmol) in DMF (15 ml) at room temperature for 18 h. DMF was removed under reduced pressure. The residue was dissolved in 70 ml ethyl acetate and washed with 50 ml water and 50 ml saturated brine. The organic extract was dried over Na 2 SO 4 and evaporated to give Fmoc silyl 83 as a sticky foam, which was treated with 20% piperidine in 70 ml DMF at ambient temperature for 8 h.
• Hydroxyl-quencher 81 is prepared from a mixture of amino-DMT compound 78 (2.5 g, 5.39 mmol) and PFP ester 79 (4.07 g, 5.90 mmol) in DCM (60 ml), treated with triethylamine (1.09 g, 10.8 mmol) at ambient temperature for 16 h (overnight). PFP ester 79 was prepared from acid 34 by the procedure of Examples 22 and 40. The mixture was concentrated and applied to a silica gel column. The column was eluted with a 0-5% methanol in DCM containing 1% triethylamine . Fractions containing products were combined and evaporated to give hydroxyl-quencher 81 as a purple foam (4.65 g, 90%).
• Quencher support 65 (Example 46, 8 mg, 200 nmoles) was loaded into a standard synthesis column and used to synthesize a T 5 oligonucleotide on an ABI 394 Synthesizer (Applied Biosystems, Foster City, Calif.) by five successive couplings of thymidine 3′ cyanoethylphosphoramidite with the standard reagents, conditions, and cycle recommended and provided by the manufacturer (Applied Biosystems) to give about 8 odu (A260 nm) of approximately 58% pure, 3′ quencher T 5 on CPG support 67:
• F is a reporter dye and Q is a quencher moiety
• Q is a quencher moiety
• the dual-labelled probes were cleaved from the CPG support, and the nucleobase and phosphate protecting groups were removed, with concentrated NH 4 OH. Deprotection can also be accomplished with a mixture of tert-butylamine/methanol/water: 1/1/2. The crude probes were analyzed and purified by reverse phase HPLC.
• PCR amplification reactions contained cDNA template nucleic acid from Raji cell RNA, 2 ⁇ TaqMan universal Master mix (25 ⁇ l) including PCR buffer, dNTPs (dATP, dGTP, dCTP, TTP), and MgCl 2 (Applied Biosystems), AmpliTaq Gold DNA polymerase, forward (SEQ ID NO: 2) and reverse (SEQ ID NO: 3) primers: Forward Primer: 5′ GGG AAG GTG AAG GTC GGA GTC 3′ SEQ ID NO:2 Reverse Primer: 5′ CTG GAA GAT GGT GAT GGG ATT TC 3′ SEQ ID NO:3
• a procedure for labelling a polypeptide, where the polypeptide is a relatively dilute protein (1 mg ml ⁇ 1 ), with a quencher-NHS ester is illustrated by the steps:
• a size-exclusion gel media column e.g. SephadexTM G10, of volume greater than 10 times the reaction volume in a disposable column. Equilibrate in 50 mM Tris-HCI, pH 7.5, or running buffer of choice. If insolubility problems have been encountered, there is a possibility of the product precipitating on the column. Experiment to find a suitable buffer: water may be a good choice.

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