Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
  • C07C245/02—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides
  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • 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
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

• This invention pertains to compounds and methods for labeling oligonucleotides.
• the invention also provides kits that contain at least one of the disclosed compounds.
• Oligonucleotides are often modified or labeled with reporter moieties such as quenchers, fluorophores, biotin, etc. These labeled oligonucleotides can provide information regarding binding and other biological phenomena, the structure of DNA, the association of macromolecules, and the size and mobility of protein and DNA complexes.
• reporter moieties such as quenchers, fluorophores, biotin, etc.
• These labeled oligonucleotides can provide information regarding binding and other biological phenomena, the structure of DNA, the association of macromolecules, and the size and mobility of protein and DNA complexes.
• Several attachment chemistries are currently used for modifying oligonucleotides. For example, primary amino groups are widely used to attach modifiers, reporter moieties or labels to an oligonucleotide. In addition, they can be used to attach an oligonucleotide to a solid surface.
• Labeled oligonucleotides have a wide variety of useful applications. For example, light quenching processes that rely on the interaction of a fluorophore and quencher as their spatial relationship changes can be used in convenient processes for detecting and/or identifying oligonucleotides and other biological phenomena.
• the change in fluorescence of a fluorophore or quencher can be monitored as two oligonucleotides (one containing a fluorophore and one containing a quencher) hybridize to each other. The hybridization can be detected without intervening purification steps that separate unhybridized from hybridized oligonucleotides.
• quencher groups are commonly placed at the end of a probe sequence while the fluorophore is placed at the opposite end, solely for ease of synthesis.
• dual-labeled probes are more effective when the labels are placed closer to each other.
• FRET fluorescent resonance energy transfer
• a fluorophore and a fluorescent quencher must be within a suitable distance for the quencher to absorb energy from the donor.
• the quencher known as BHQ-I which absorbs light in the wavelength range of about 520-550 nm, can quench the fluorescent light emitted from the fluorophore, fluorescein, which fluoresces maximally at about 520 nm.
• a PCR reaction is performed using oligonucleotides designed to hybridize to the 3' side ("downstream") of an amplification primer so that the 5'-3' exonuclease activity of a polymerase digests the 5' end of the probe, cleaving off one of the dyes.
• the fluorescence intensity of the sample increases and can be monitored as the probe is digested during the course of amplification.
• Similar oligonucleotide compositions may be used in other molecular/cellular biology and diagnostic assays, such as end-point PCR, in situ hybridizations, in vivo DNA and RNA species detection, single nucleotide polymorphism (SNPs) analysis, enzyme assays, and in vivo and in vitro whole cell assays.
• end-point PCR in situ hybridizations
• in vivo DNA and RNA species detection in vivo DNA and RNA species detection
• SNPs single nucleotide polymorphism
• enzyme assays enzyme assays
• in vivo and in vitro whole cell assays in vivo and in vitro whole cell assays.
• the invention provides a method for linking a reporter moiety to an oligonucleotide comprising reacting a reporter moiety having an oxime forming nucleophile substituent with an oxo substituted reactant coupled to a solid support to form an oxime bond between the reporter moiety and the reactant.
• the reporter moieties include, but are not limited to, quenchers, fluorophores, biotin, digoxigenin, peptides and proteins.
• the invention also provides an oligonucleotide labeled with at least two different reporter moieties.
• This invention further provides novel azo quenchers having the general formula shown below in Formula (I):
• Each OfR 1-6 is individually selected from the group consisting of hydrogen; electron withdrawing groups such as halogens, NO 2 , SO 3 Rs, SO 2 N(RN) 2 , CN, CNS, keto, alkoxy groups; C 1 -C 10 alkyl groups; aryl groups; and heteroaryl groups.
• RN and Rs can be C 1 -C 10 alkyl groups, which may be saturated or unsaturated, branched or unbranched, and substituted or unsubstituted, or aryl groups, which may be substituted or unsubstituted.
• Suitable substituents include electron withdrawing groups, such as those described above.
• R 7 can be any aryl group that can be joined to the conjugated ring system by an azo bond to form a compound that is capable of quenching the fluorescence of a fluorophore.
• Suitable aryl groups include phenyl, naphthyl, benzyl, xylyl, toluyl, pyridyl and anilinyl, among other groups.
• R 7 can be substituted or derivatized with at least one linking group for linking the quencher compound to other compounds of interest.
• Y is a nucleophile-containing group capable of reacting with an oxo group to form an oxime bond, such as aminooxy or hydrazine.
• R 1 ZR 2 pair, R 3 ZR 4 pair, R 4 ZR 5 pair and R 5 ZR 6 pair can be combined to form ring structures having five or six ring members.
• ring structures can be substituted with hydrogen, heteroatom-substituted alkyl, halogen, alkenyl, alkoxy, alkoxy-alkyl, hydroxyl, trifluoromethyl, cyano, nitro, acyl, acyloxy, amino, alkylamino, dialkylamino, carboxyl, carbalkoxyl, carboxamido, mercapto, sulfamoyl, phenyl, and napthyl.
• this invention provides an oligonucleotide labeled with the novel quencher as well as a method of detecting hybridization of oligonucleotides using the labeled oligonucleotide.
• the invention provides compositions comprising a quencher linked to a compound selected from the group consisting of an antigen, a steroid, a vitamin, a drug, a hapten, a metabolite, a toxin, an environmental pollutant, an amino acid, a protein, a carbohydrate, a solid support, a linker, and a lipid, wherein the quencher is attached to the compound via an oxime bond.
• the invention further provides compositions comprising labeled oligonucleotides and solid supports.
• the invention also provides kits comprising at least one composition of the present invention.
• FIG. 1 shows the synthesis of a compound of Formula (I).
• FIG. 2 shows the synthesis of a ketone phosphoramidite.
• FIG. 3 shows the synthesis of aminooxy conjugated controlled pore glass supports.
• FIG. 4 shows the introduction of the aminooxy group into a reporter moiety that is stable to basic conditions.
• FIG. 5 shows the introduction of the aminooxy group into a base labile reporter moiety.
• FIG. 6 shows real-time PCR data for Probe SEQ ID NO: 1 in a multicomponent view.
• Fluorescein data plot is positioned as the first curve in the upper graph and represents signal from the probe.
• Rox data plot is positioned as the second (flat) curve in the upper plot and represents detection control. Temperature trace during thermal cycling is plotted in the lower graph.
• FIG. 7 shows real-time PCR data for Probe SEQ ID NO: 1 as amplification traces. Reactions were done using input target amounts of 5 x 10 6 molecules, 5 x 10 4 molecules, and 5 x 10 2 molecules which are shown left to right. All target concentrations were run in triplicate.
• FIG. 8 shows real-time PCR amplification traces for Probe SEQ ID NO: 2. Reactions were done using input target amounts of 5 x 10 molecules, 5 x 10 4 molecules, and 5 x 10 2 molecules which are shown left to right. All target concentrations were run in triplicate.
• FIG. 9 shows real-time PCR amplification traces for Probe SEQ ID NO: 3.
• FIG. 10 shows real-time PCR amplification traces for Probe SEQ ID NO: 4. Reactions were done using input target amounts of 5 x 10 molecules, 5 x 10 4 molecules, and 5 x 10 2 molecules which are shown left to right. AU target concentrations were run in triplicate.
• FIG. 11 shows real-time PCR amplification traces for Probe SEQ ID NOS : 1 -4. Traces for each probe using 5 x 10 6 input target molecules are shown. All target concentrations were run in triplicate.
• FIG. 12 shows real-time PCR amplification traces for probe SEQ ID NOS: 11-14.
• FIG. 13 shows the absorbance spectrum of an oligonucleotide of SEQ ID NO: 15.
• FIG. 14 shows the synthesis of a fluorescein aminooxy derivative.
• FIG. 15 shows examples of aminooxy substituted reporter moieties.
• the invention provides a novel method of labeling oligonucleotides with reporter moieties during synthesis of the oligonucleotide.
• the method permits the attachment of several different reporter moieties to a single oligonucleotide.
• reporter moiety refers to a substituent that allows detection, either directly or indirectly, of a compound at low concentrations.
• Suitable reporter moieties include, but are not limited to, (1) enzymes, which produce a signal detectable, for example, by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase or glucose-6-phosphate dehydrogenase; (2) chromophores, such as fluorescent, luminescent or dye compounds; (3) groups with an electron density which can be detected by electron microscopy or through their electrical property, such as by conductivity, amperometry, voltametry, or impedance measurements; and (4) groups which can be detected using optical methods, such as diffraction, surface plasma resonance or contact angle variation, or physical methods, such as atomic force spectroscopy, or the tunnel effect.
• Other suitable reporter moieties include, but are not limited to, biotin, dig
• the method comprises forming an O-substituted oxime ("oxime") bond between a reporter moiety having a nucleophile capable of forming an oxime bond with an oxo group (also referred to as a nucleophile containing reporter moiety) and an oxo-substituted reactant.
• oxime bond is completely orthogonal to reactions during phosphoramidite oligonucleotide synthetic cycle and can be used as a universal method for introduction of multiple modifications into an oligonucleotide.
• the oxime bond may be used to introduce almost any modification into an oligonucleotide during synthesis or prior to synthesis by modification of the solid support.
• the bond is unexpectedly stable, and remains intact during thermocycling. This method also permits the introduction of multiple different reporter moieties into an oligonucleotide.
• the oxo-substituted reactant can be an oxo-substituted oligonucleotide which is linked to a solid support, an oxo-substituted nucleotide, an oxo-substituted nucleoside, an oxo- substituted nucleoside phosphoramidite, or a composition of Formula (II):
• R is H or alkyl
• PG is a hydroxyl protecting group, such as those commonly used in oligonucleotide synthesis, e.g. dimethoxytrityl (DMT), monomethoxytrityl (MMT), or trityl
• A is a linker used to attach an oligonucleotide to a solid support during synthesis of the oligonucleotide, such as the phosphate linkers, shown in 20a and 20b of FIG. 3.
• the alkyl is selected from a C 1-6 alkyl group, which is substituted or unsubstituted, branched or unbranched, and saturated or unsaturated.
• Suitable substituents include, but are not limited to, alkoxy, hydroxyl, cyano, amino, alkylamino, dialkylamino, halogen, alkylthio, and thiol.
• the oxo-substituted nucleotide and oxo-substituted nucleoside can be attached to a solid support.
• the oxo-substituted oligonucleotides, oxo-substituted nucleotides, oxo-substituted nucleosides, and oxo-substituted nucleoside phosphoramidites for use in the present invention include those containing the traditional nucleobases, such as adenine, guanine, cytosine, uracil and thymine, and those containing modified nucleobases.
• solid support refers to any support that is compatible with oligonucleotide synthesis.
• the following are suitable: glass, controlled pore glass, polymeric materials, polystyrene beads, coated glass, and the like.
• the method permits incorporation of an oxo-substituted nucleotide into an oligonucleotide followed by reaction with a reporter moiety having a nucleophilic substituent capable of forming an oxime bond with the oxo group.
• the reporter moiety can be added immediately after the oxo-substituted nucleotide is added to the oligonucleotide or the reporter moiety can be added after additional nucleotides or oxo- substituted nucleotides have been added to the oligonucleotide.
• the novel method permits internal incorporation of a reporter moiety into an oligonucleotide as a reporter moiety substituted nucleotide which is incorporated into the oligonucleotide using standard phosphoramidite chemistry.
• the nucleophile containing reporter moiety can be reacted with an oxo-substituted reactant.
• the resulting composition, a reporter moiety substituted reactant is then used to derivatize a solid support, as in Example 3, and the derivatized support can serve as the foundation for oligonucleotide synthesis by standard methods.
• Example 3 demonstrates the attachment of an azo quencher compound to controlled pore glass, the method is more generally applicable to the attachment of a reporter moiety to any solid support that contains free reactive electrophile groups, including ketones and aldehydes.
• the solid support bound reporter moiety can be used conveniently in conjunction with automated oligonucleotide synthesizers to directly incorporate the reporter moiety into oligonucleotides during their synthesis.
• the present method allows for multiple reporter moieties to be introduced into a single oligonucleotide.
• the reporter moieties may be the same or different.
• Use of different reporter moieties on a single oligonucleotide allows detection of multiple signals using a single oligonucleotide. Detection may be simultaneous or sequential.
• the invention also provides novel azo compounds that are useful as fluorescence quenchers.
• the quenchers of this invention which release energy absorbed from fluorophores without emitting light, i.e. are "dark quenchers", have the general formula shown below in Formula (I).
• Each OfR 1-6 is individually selected from the group consisting of hydrogen, electron withdrawing groups such as halogens, NO 2 , SO 3 Rs, SO 2 N(RN) 2 , CN, CNS, keto, and alkoxy groups, C 1 -C 1O alkyl groups, aryl groups, and heteroaryl groups.
• RN and Rs can be C 1 -C 10 alkyl groups, which may be branched or unbranched and saturated or unsaturated, and substituted or unsubstituted, and aryl groups, which may be substituted or unsubstituted.
• Suitable substituents include electron withdrawing groups such as those described above.
• R 7 can be any aryl group that can be joined to the conjugated ring system by an azo bond to form a compound that is capable of quenching the fluorescence of a fluorophore.
• Suitable aryl groups include phenyl, naphthyl, benzyl, xylyl, toluyl, pyridyl, and anilinyl, among other groups.
• R 7 can be substituted or derivatized with at least one linking group for linking the quencher compound to other compounds of interest.
• Y is a nucleophile-containing group capable of reacting with an oxo group to form an oxime bond, such as aminooxy or hydrazine.
• an oxo group such as aminooxy or hydrazine.
• R 4 ZR 5 pair and R 5 /R 6 pair can be combined to form ring structures having five or six ring members.
• These ring structures can be substituted with hydrogen, heteroatom-substituted alkyl, halogen, alkenyl, alkoxy, alkoxy-alkyl, hydroxyl, trifluoromethyl, cyano, nitro, acyl, acyloxy, amino, alkylamino, dialkylamino, carboxyl, carbalkoxyl, carboxamido, mercapto, sulfamoyl, phenyl, and napthyl.
• reactive substituents at R 1-6 such as amino, hydroxyl, and carboxyl groups, can be attached to linking groups or other molecules of interest.
• linking group refers to a chemical group that is capable of reacting with a "complementary functionality" of a reagent, e.g., to the ketone group of a phosphoramidite, to form a bond that connects the azo quenching compound of
• Fluorescent Probes and Research Chemicals, Molecular Probes, Inc. disclosing numerous modes for conjugating a variety of dyes to a variety of compounds, which is incorporated herein by reference.
• R 7 -Y is the compound of Formula (III) where the aryl ring is an anilinyl group which can be substituted with various groups at positions L and L'.
• L and L' are independently selected from the group consisting of substituted or unsubstituted C 1- 10 alkyl and nucleophile-containing C 1-10 alkyl groups, wherein the C 1-10 alkyl groups are saturated or unsaturated.
• one of L or L' can be a nonreactive group (i.e., one that does not contain a nucleophile and cannot be modified to contain a nucleophile), such as an alkyl group, preferably an ethyl group, and the other can be a reactive group, such as a hydroxyethyl group which can be modified further to a nucleophilic group such as aminooxy to facilitate linking the quencher to other molecules of interest.
• hydroxy alkyl chains of any length could be used to modify the anilinyl group.
• the azo quencher compound has the structure of Formula (V), wherein Y is an aminooxy group.
• Suitable azo quencher precursor compounds have a primary amino group and have the general structure of Formula (VI).
• Specific embodiments of Formula (VI) include compounds 1 and 2.
• the azo quenchers of Formula (I) are suitable for incorporation into oligonucleotides as is discussed above.
• the azo quenchers of Formula (I) can be linked to a variety of other useful compounds, provided that suitable reactive groups are present on those compounds.
• Such compounds include antigens, antibodies, steroids, vitamins, drugs, haptens, metabolites, toxins, environmental pollutants, amino acids, proteins, carbohydrates, lipids, and the like.
• Examples of other aminooxy substituted reporter moieties are shown in Figure 15.
• the invention also is directed to oligonucleotide compositions containing dye pairs, which include one of the disclosed quencher compounds and a fluorophore that fluoresces on exposure to light of the appropriate wavelength.
• Suitable fluorophores in the dye pair are those that emit fluorescence that can be quenched by the quencher of the dye pair.
• the dye pair can be attached to a single compound, such as an oligonucleotide. Li other embodiments, the fluorophore and the quencher can be on different compounds. [0053] A wide variety of reactive fluorophores are known in the literature and can be used with a corresponding quencher.
• the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound.
• Suitable fluorophores include xanthene dyes, such as fluorescein or rhodamine dyes, including 6-carboxyfluorescein (FAM) 3 2'7'-dimethoxy- 4'5'-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX).
• Suitable fluorophores also include the naphthylamine dyes that have an amino group in the alpha or beta position.
• naphthylamino compounds include l-dimethylaminonaphthyl-5- sulfonate, l-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate, 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS).
• fluorophores include coumarins, such as 3-phenyl-7-isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-( - carboxy-penty ⁇ -S'-ethyl-S ⁇ '-dimethyloxacarbocyanine (CyA); 1H,5H,1 IH 5 15H- Xantheno[2,3,4-ij:5,6,7-i'j']diquinolizin- 18-ium, 9-[2(or 4)-[[[6-[2,5-dioxo-l-pyrrolidinyl)oxy]- 6- ox
• 6-Carboxyfluorescein (6-FAM) 520 nm
• TET Tetrachlorofluorescein
• TAMRA Tetramethylrhodamine
• the quencher of Formula (I) is capable of absorbing the fluorescent energy in the range of about 500 to about 620 nm and therefore can be used to quench the fluorescence of fluorescein through Texas Red.
• fluorophores are available and can be used depending on the circumstances.
• substituents can be attached to xanthene rings for bonding with various reagents, such as for bonding to oligonucleotides.
• fluorescein and rhodamine dyes appropriate linking methodologies for attachment to oligonucleotides have also been described. See, for example, Khanna et al. U.S. Patent 4,439,356, which is incorporated herein by reference; Marshall (1975) Histochemical J., 7:299-303, which is incorporated herein by reference; Menchen et al., U.S.
• Patent 5,188,934 which is incorporated herein by reference; Menchen et al., European Patent Application No. 87310256.0, which is incorporated herein by reference; and Bergot et al., International Application PCT/U590/05565, which is incorporated herein by reference.
• Other quenchers could potentially be incorporated into an oligonucleotide using the method of the present invention. Some of these are shown in Table 1 below.
• the dye pair when the dye pair is in a configuration in which fluorophore is effectively quenched by the quencher dye, its fluorescence is reduced by at least a factor of 80%, and more preferably by 90%, 95%, or 98%, when compared to its fluorescence in the absence of quenching.
• High levels of quenching allow for the preparation of oligonucleotide probes having a high signal to noise ratio which is defined as the amount of signal present when the composition is in its maximal unquenched state (signal) versus its maximally quenched state (noise).
• Probes having a high signal to noise ratio are desirable for the development of highly sensitive assays.
• To measure signal to noise ratios relative fluorescence is measured in a configuration where the quencher and fluorophore are within the F ⁇ rster distance and the fluorophore is maximally quenched (background fluorescence or "noise") and compared with the fluorescence measured when fluorophore and quencher are separated in the absence of quenching ("signal").
• the signal to noise ratio of a dye pair of the invention will generally be at least about 2:1 but generally is higher. Signal to noise ratios are generally affected by the fluorophore-quencher pair, the quality of the synthesis, and the oligonucleotide sequence.
• Oligonucleotide probes that include a dye pair can be used to detect target oligonucleotides.
• the individual components of a dye pair can be on opposing, hybridizable, self-complementary segments of a single oligonucleotide such that when the oligonucleotide hybridizes to itself in the absence of exogenous sequences, FRET occurs.
• the oligonucleotide probe is constructed in such a way that the internal hybridizing is disrupted and fluorescence can be observed when the oligonucleotide probe hybridizes to a complementary target oligonucleotide.
• oligonucleotide probe can be used to rapidly detect target oligonucleotides having sequences that bind to the oligonucleotide probe.
• a composition comprises two biomolecules, such as oligonucleotides, with a fluorophore attached to one of the biomolecules and a quencher attached to the other.
• Oligonucleotide probes lacking self-complementarity can also be utilized in a similar manner.
• a quencher and fluorophore can be placed on an oligonucleotide that lacks the self-hybridizing property such that the random-coil conformation of the oligonucleotide keeps the fluorophore and quencher within a suitable distance for fluorescence quenching.
• oligonucleotides can be designed so that when they hybridize to desired target oligonucleotides the fluorophore and quencher are further apart and fluorescence can be observed.
• oligonucleotide probes can be designed such that they can hybridize adjacent to each other on a contiguous length of a target oligonucleotide.
• the two probes can be designed such that when they are hybridized to the target oligonucleotide, a quencher on one of the oligonucleotide probes is within a sufficient proximity to a fluorophore on the other oligonucleotide probe for FRET to occur.
• Binding of the oligonucleotide probes to the target oligonucleotide can be followed as a decrease in the fluorescence of the fluorophore.
• a set of oligonucleotides that hybridize to each other can be configured such that a quencher and a fluorophore are positioned within the F ⁇ rster distance on opposing oligonucleotides. Incubation of such an oligonucleotide duplex with another oligonucleotide that competes for binding of one or both of the oligonucleotides would cause a net separation of the oligonucleotide duplex leading to an increase in the fluorescent signal of the fluorophore. To favor binding to the polymer strands, one of the oligonucleotides could be longer or mismatches could be incorporated within the oligonucleotide duplex.
• the assay utilizes an oligonucleotide that is labeled with a fluorophore and a quencher in a configuration such that fluorescence is substantially quenched.
• the oligonucleotide is designed to have sufficient complementarity to a region of the amplified oligonucleotide so that it will specifically hybridize to the amplified product.
• the hybridized oligonucleotide is degraded by the exonuclease activity of Taq polymerase in the subsequent round of DNA synthesis.
• the oligonucleotide is designed such that as the oligomer is degraded, one of the members of the dye pair is released and fluorescence from the fluorophore can be observed. An increase in fluorescence intensity of the sample indicates the accumulation of amplified product.
• Ribonucleic acid polymers can also be configured with fluorophores and quenchers and used to detect RNase.
• a dye pair can be positioned on opposite sides of an RNase cleavage site in an RNase substrate such that the fluorescence of the fluorophore is quenched.
• Suitable substrates include oligonucleotides that have a single-stranded region that can be cleaved and that have at least one internucleotide linkage immediately 3' to an adenosine residue, at least one internucleotide linkage immediately 3' to a cytosine residue, at least one internucleotide linkage immediately 3' to a guanosine residue and at least one internucleotide linkage next to a uridine residue and optionally can lack a deoxyribonuclease-cleavable internucleotide linkage.
• the substrate can be incubated with a test sample for a time sufficient for cleavage of the substrate by a ribonuclease enzyme, if present in the sample.
• the substrate can be a single-stranded oligonucleotide containing at least one ribonucleotide residue at an internal position.
• the fluorescence of the fluorophore whose emission was quenched by the quencher, becomes detectable. The appearance of fluorescence indicates that a ribonuclease cleavage event has occurred, and, therefore, the sample contains ribonuclease activity.
• kits that comprise a labeled oligonucleotide or an azo quencher of the present invention.
• the kit can also contain instructions for use.
• Such kits can be useful for practicing the described methods or to provide materials for synthesis of the compositions as described. Additional components can be included in the kit depending on the needs of a particular method. For example, where the kit is directed to measuring the progress of PCR reactions, it can include a DNA polymerase. Where a kit is intended for the practice of the RNase detection assays, RNase-free water could be included. Kits can also contain negative and/or positive controls and buffers.
• N-Fmoc-3-aminopropyl solketal (10) 3-Aminopropyl solketal (9) was synthesized starting from commercially available solketal (7) according to the procedure of Misiura et al (Misiura, K., Durrant, L, Evans, M.R., Gait, MJ. (1990) Nucleic Acids Research, v. 18, No. 15, pp. 4345-4354, which is incorporated herein by reference). (9) was used crude without vacuum distillation for the next step. The crude product (9) (12.85 g; 68 mmol) was dissolved in dry CH 3 CN (100 mL) with stirring.
• the product (10) was isolated by flash chromatography on a silica gel column (5x20 cm) loading from EtOAc: CH 2 Cl 2 :petroleum ether (PE) (15:15:70) and eluting with EtOAc: CH 2 Cl 2 :PE (1:1:2).
• the isolated product (10) had R f of 0.4 by TLC in EtOAc:CH 2 Cl 2 :PE (1:1:1). Yield: 20.95 g of oil.
• l-0-DMT-3-0-(N-Fmoc-3-aminopropynglvcerol (12) l-O-(N-Fmoc-3- aminopropyl) glycerol (11) (2.64 g; 7.1 mmol) was dissolved in dry pyridine (50 mL) and treated with DMT-Cl (2.65 g; 7.8 mmol). The reaction mixture was stirred at room temperature overnight and quenched with MeOH (5 mL). It was then concentrated to oil under reduced pressure. The residue was dissolved in EtOAc ( ⁇ 300 mL) and extracted with saturated NaHCO 3 (3x100 mL) followed by brine (100 mL).
• Pentafluorophenyl 5-oxohexanoate (14) 5-Oxohexanoic acid (2.6 g; 20 mmol) was dissolved in CH 2 Cl 2 (50 mL). N, N-Diisopropylethylamine (10.4 mL, 60 mmol) was added followed by pentafluorophenyl trifluoroacetate (3.61 mL; 21 mmol). The reaction mixture was kept at room temperature for 1 hour and evaporated. The residue was resuspended in EtOAc:Hexanes (1:1) and loaded on a silica gel column (5x20 cm) equilibrated and developed with the same mixture.
• the solid support was treated with 0.1 M I 2 in THF/Py/H 2 O (3x30 mL; 5 minutes each treatment), and washed with CH 3 CN (5x30 mL). Unreacted hydroxyls were capped by treating with Ac 2 O:MeIm:Py (10:10:80) (3x30 mL; 5 minutes each treatment).
• the derivatized CPG (19a) was washed with CH 3 CN (5x30 mL), CH 2 Cl 2 (3x30 mL), and dried in vacuum overnight. DMT-loading was usually above 30 ⁇ mol/g.
• S :N signal to noise ratio
• oligonucleotides containing both fluorescein and the azo quencher as prepared in Examples 1 through 3.
• Fluorescence-quenched probes are employed in a variety of applications in molecular biology.
• One method to assess if a given fluorophore and a quencher function well together is by measurement of a signal to noise ratio (S :N), where relative fluorescence is measured in the native configuration (background fluorescence or "noise") and compared with fluorescence measured when fluorophore and quencher are separated (“signal").
• Oligonucleotide Synthesis The following oligonucleotides were synthesized using standard phosphoramidite chemistry and the aminooxy quencher reagents described in Example 3, supra. Oligonucleotides were purified by HPLC. Dual-labeled oligonucleotides were made with the novel aminooxy (l-nitro-4-naphthylazo)- ⁇ iV-diethanolaniline quencher (6) of the invention at the 3 '-end of the probe with the fluorescein reporter group placed at the 5 'end (6- FAM 5 single isomer 6-carboxyfluorescein, Glen Research, Sterling, VA).
• FAM-oxime conjugate has to be acetylated with acetic anhydride capping reagent prior following phosphoramidite cycle.
• Electrospray-ionization liquid chromatography mass spectroscopy (ESI-LCMS) of each oligonucleotide probe was performed using an Oligo HTCS system (Novatia, Princeton, NJ), which consisted of ThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processing software and Paradigm MS4TM HPLC (Michrom BioResources, Auburn, CA). Protocols recommended by manufacturers were followed. Experimental molar masses for all compounds were within 0.02% of expected molar mass, confirming the identity of the compounds synthesized.
• Oligonucleotides were evaluated for quenching efficiency in a pre- and post-nuclease degradation assay.
• Probe oligonucleotides (SEQ ID NOS: 1-4) were individually resuspended at 100 iiM concentration in HPLC-grade water. From this stock solution, 2 ml of 100 nM probe solution was prepared with STNR Buffer, comprising 10 niM Tris pH 8.3, 50 mM KCl, 5 mM MgCl 2 , 1 mM CaCl 2 , which was split into two identical 1 niL fractions. One fraction was retained without enzyme treatment as background control. The second fraction was subjected to nuclease degradation as follows.
• Micrococcal nuclease 15 units (Roche, 15U/ul), was added to the oligonucleotide solution and incubated at 37 0 C for 1 hour. Relative fluorescence intensity for each sample was measured with a PTI QuantaMaster Model C-60 cuvette-based spectrofluorometer (Photon Technology International, Monmouth Jet., NJ). The fluorescence measurement of the solution containing intact probe constituted the "background” or "noise” component of the assay. The fluorescence measurement of the solution containing degraded probe (nuclease treated) constituted the "signal" component of the assay. Signal to noise ratios (S :N) were calculated. Table 3: Signal to Noise ratios for Fluorescence-Quenched Linear Oligonucleotides.
• the novel aminooxy attached quenchers (6) are capable of quenching a fluorescein with similar efficiency as a commonly employed commercially available quencher group.
• Fluorescence-quenched probes can be employed to detect a target nucleic acid sequence. Commonly, such detection is linked to an amplification step, such as the polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• Oligonucleotide primers were synthesized using standard phosphoramidite chemistry, desalted, and employed without additional purification. Probe oligonucleotides employed are the same compounds studied in Example 4 supra, SEQ ID NOS: 1-4. Primers employed are:
• the target nucleic acid is SEQ ID NO: 7, a 150 base pair (bp) aniplicon derived from the human bHLH protein PTFlA gene (Genbank # NMJ78161), cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), and is hereafter referred to as the "p48-gene target”.
• Target Nucleic Acid Sequence a 150 base pair (bp) aniplicon derived from the human bHLH protein PTFlA gene (Genbank # NMJ78161), cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), and is hereafter referred to as the "p48-gene target”.
• PCR amplification was done using the thermostable DNA polymerase ImmolaseTM (Bioline, Randolph, MA), 800 uM dNTPs, and 3 mM MgCl 2 . Reactions were carried out in a 25 ⁇ L volume and comprised 200 nM each of the amplification primers and fluorescent quenched probe, 500, 50,000 and 5,000,000 copies of target DNA. Cycling conditions were 50° C for 2 min, 95° C for 10 min, then 40 cycles of 2-step PCR with 95° C for 15 sec and 60° C for 1 min. PCR and fluorescence measurements were done using an ABI PrismTM 7700 Sequence Detector (Applied Biosystems Inc., Foster City, CA). All data points were performed in triplicate.
• the cycle threshold (Ct) value is defined as the cycle at which a statistically significant increase in fluorescence is detected above background. A lower Ct value is indicative of a higher concentration of target DNA.
• the assays were performed using an identical amount of input target DNA (5 x 10 2 -5xl0 4 -5xl0 6 copies of the PTFIa p48-gene target plasmid). Relative fluorescence levels collected during PCR for each probe were graphically plotted against cycle number and are shown in FIGS. 1-5.
• FIG. 6 The multicomponent view of a 40-cycle real-time PCR reaction using probe SEQ ID NO: 1, which incorporates the aminooxy (l-nitro-4-naphthylazo)-iV,iV-diethanolanilme quencher (6) attached post-synthetically (IBAOket), is shown in FIG. 6.
• the fluorescence baseline remained flat until cycle 18, when product first reached detectable levels as a result of PCR amplification.
• the oxime bond was stable in the employed reaction conditions and no elevation of baseline background fluorescence was observed. The oxime bond is therefore suitable for use in these reaction conditions, which are similar to conditions commonly employed in many molecular biology applications.
• Amplification traces for probe SEQ ID NO: 3, which incorporates the aminooxy (1 - nitro-4-naphthylazo)-iV ) N-diethanolaniline quencher attached during synthesis as a CPG conjugate via direct linkage to the 3 '-end of the oligonucleotide (IBAOC7), are shown in FIG. 8. The results showed good clustering of triplicate reactions and clearly distinguished between different input concentrations of the target nucleic acid.
• Amplification traces for probe SEQ ID NO: 4, which incorporates commercial Eclipse Quencher attached during synthesis as a CPG conjugate via direct linkage to the 3 '-end of the oligonucleotide (Eclipse), are shown in FIG. 9. The results showed good clustering of triplicate reactions and clearly distinguished between different input concentrations of the target nucleic acid.
• Table 4 summarizes the real-time PCR results and demonstrates that all oligonucleotides provided similar Ct values regardless of method of quencher attachment and functioned with similar performance in this application.
• probe compositions comprising the new aminooxy (l-nitro-4- naphthylazo)-iV,N-diethanolaniline quencher (6)of the invention performed well in a quantitative real-time PCR assay and were functionally interchangeable with probes that contain other quencher moieties.
• Quencher groups are commonly placed at the end of a probe sequence for ease of synthesis.
• the new aminooxy quencher permits internal incorporation of quencher as a base modified aminooxy quencher-dU moiety.
• This example demonstrates that use of fluorescent probes modified with internal aminooxy-quenchers function better in a real-time PCR assay than standard end-quenched probes.
• Dual-labeled oligonucleotides with internal modifications were made using ketone-dU phosphoramidite (synthesized according to published procedure: Dey & Shepard, (2001) Org. Lett., v.3, pp. 3983-3986, which is incorporated by reference herein) followed by interconjugation with 300 ⁇ L of 10 mM solution (per 1 ⁇ mole of the oligonucleotide on the solid supprt) of the aminooxy-quencher reagent (6) in ethanol at the time of synthesis. After 2 hours, excess aminooxy-quencher was removed, the solid support was washed with 1 mL of acetonitrile and the oligonucleotide was extended using standard phosphoramidite chemistry.
• Oligonucleotide primers were synthesized using standard phosphoramidite chemistry, desalted, and were used in the assay without additional purification. Primer and probe oligonucleotides employed are shown below. Probes with internal quencher modifications had a C3 spacer group placed at the 3 '-end in place of the quencher group to block extension during PCR. Oligonucleotides were synthesized as described above.
• SEQ ID NOS: 8 and 9 Internal aminooxy-quencher-dU is notated by (ilBAOdU).
• SEQ ID NO: 11 represents a traditional probe with 3 '-terminal quencher placement.
• SEQ ID NO: 12 has an internal aminooxy-quencher-dU substitution for an internal dT base at position 9 from the 5 '-end.
• SEQ ID NO: 13 has an internal aminooxy-quencher-dU substitution for an internal dC base at position 10 from the 5'- end, which results in a favorable U:G base pairing event upon hybridization.
• SEQ ID NO: 14 has an internal aminooxy-quencher-dU substitution for an internal dA base at position 13 from the 5 '-end, which results in an unfavorable U:T base pairing event upon hybridization.
• the aminooxy-quencher-dU base is compound (20a_.
• the target nucleic acid is SEQ ID NO: 10, a 162 base pair (bp) amplicon derived from the human Enolase gene (Genbank # NM_001428), cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), and is hereafter referred to as the "hEnolase-gene target”.
• Target Nucleic Acid Sequence SEQ ID NO: 10
• PCR amplification was performed using the thermostable DNA polymerase ImmolaseTM (Bioline, Randolph, MA), 800 uM dNTPs, and 3 mM MgCl 2 . Reactions were carried out in a 25 ⁇ L volume and comprised 200 nM each of the amplification primers and fluorescent quenched probe, 500, 50,000 and 5,000,000 copies of target DNA. Cycling conditions were 50° C for 2 min, 95° C for 10 min, then 40 cycles of 2-step PCR with 95° C for 15 sec and 65° C for 1 min. PCR and fluorescence measurements were done using an ABI PrismTM 7700 Sequence Detector (Applied Biosystems Inc., Foster City, CA). All data points were performed in triplicate.
• the cycle threshold (Ct) value is defined as the cycle at which a statistically significant increase in fluorescence is detected above background. A lower Ct value is indicative of a higher concentration of target DNA.
• the assays were performed using an identical amount of input target DNA (5 x 10 2 -5xl0 4 -5xl0 6 copies of the hEnolase-gene target plasmid). Relative fluorescence levels collected during PCR for each probe were graphically plotted against cycle number. The real-time PCR results for all 4 probes are plotted together for a single target concentration, 5 x 10 6 and are shown in FIG. 12. The absolute change in fluorescence ( ⁇ Rf) varied noticeably between probes.
• probes had similar quality and the differences in fluorescence relates to different potency of quenching that varies with quencher placement.
• Actual sensitivity to quantitative detection of the input target nucleic acid varied between probes and is quantified in Table 6 below. Table 6. Relative Ct Values for Probes SEQ ID NOS: 10-13 in Real Time PCR Assay.
• Probe compositions comprising the new aminooxy (l-nitro-4-naphthylazo)-N,N- diethanolaniline quencher (6) placed internally on a dU base show superior properties in a realtime PCR assay compared with standard 3 '-quencher probes. Detection limits were improved by ⁇ 1 Ct value, which corresponds to about double detection sensitivity.
• This example shows an absorbance spectrum of an oligonucleotide modified at its 5 'terminus to contain the azoquencher (6).
• the oligonucleotide was made using standard automated phosphoramidite nucleotide synthetic methods where the last addition cycle was carried out with the molecule (6).
• the composition of the oligonucleotide is shown below. SEQ ID NO: 15 (Azo-Quencher)-CAGAGTACCTGA
• the oligonucleotide was suspended in HPLC-grade water at 400 nM concentration.
• Optical absorbance was measured in 10 mM Tris pH 8.0, 1 mM EDTA (TE buffer) with a sub-micro quartz cuvette with 1-cm path length in a Hewlett Packard Model 8453 spectrophotometer (Hewlett Packard, Palo Alto, CA). Absorbance density was recorded from 220 run to 700 nm and is shown in FIG. 13.
• the absorbance spectrum was broad, ranging from 420 to 620 nm, with peak absorbance at 531 nm. This absorbance range overlaps with the fluorescence emission of a wide variety of fluorophores commonly used in molecular biology applications. For FRET based quenching mechanisms, this spectrum is positioned to offer maximum quenching capacity for dyes in the spectral range of fluorescein.
• the aminooxy group is introduced to a reporter moiety via the Mitsunobu reaction between alcohol (21) and iV-hydroxyphthalimide followed by phthalimide hydrolysis. (Scheme 4 in FIG. 4).
• This method can be used for derivatization of fluorophores, quenchers, biotin, peptides and other reporter moieties stable to basic conditions.

Priority Applications (4)

Applications Claiming Priority (2)

Publications (2)

Family

ID=37452651

Family Applications (1)

Country Status (6)

Cited By (5)

Families Citing this family (28)

Family Cites Families (105)

• 2006
  • 2006-05-22 JP JP2008512555A patent/JP2008545659A/ja active Pending
  • 2006-05-22 EP EP06770717A patent/EP1907560B1/en not_active Not-in-force
  • 2006-05-22 AU AU2006251637A patent/AU2006251637B2/en not_active Ceased
  • 2006-05-22 WO PCT/US2006/019552 patent/WO2006127507A2/en active Application Filing
  • 2006-05-22 CA CA002601554A patent/CA2601554A1/en not_active Abandoned
  • 2006-05-22 US US11/438,606 patent/US7476735B2/en active Active
• 2009
  • 2009-01-12 US US12/352,125 patent/US7605243B2/en not_active Expired - Fee Related
  • 2009-08-04 US US12/535,220 patent/US7645872B2/en not_active Expired - Fee Related
  • 2009-11-23 US US12/623,811 patent/US7803936B2/en active Active
• 2010
  • 2010-08-10 US US12/853,741 patent/US8030460B2/en not_active Expired - Fee Related
• 2011
  • 2011-09-02 US US13/224,571 patent/US8258276B2/en active Active

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events