Oligonucleotides labeled with a plurality of fluorophores

Abstract

An embodiment of the invention discloses new methods for designing labeled nucleic acid probes and primers by labeling oligonucleotides with a plurality of spectrally identical or similar dyes and optionally with one or more quencher dyes. Oligonucleotides labeled in accordance with some embodiments of the invention exhibit a detectable increase in signal, for example, fluorescent signal when the labeling dyes are separated from one another. Methods for separating the dye include cleaving the labeled oligonucleotides include using enzymes that have 5′-exonuclease activity. In one embodiment nucleic acid primers of the present invention may fluoresce upon hybridization to a target sequence and incorporation into the amplification product. Nucleic acid probes and primers of the present invention have wide applications ranging from general detection of a target nucleic acid sequence to clinical diagnostics. Major advantages of the oligonucleotides including nucleic acid probes and primers of many embodiments of the present invention are their synthetic simplicity, spectral versatility and superior fluorescent signal.

1 . A labeled oligonucleotide, comprising:
 an oligonucleotide sequence that hybridizes to a target polynucleotide sequence; and   at least two photometric labeling molecules attached to said oligonucleotide;   wherein said at least two photometric labeling molecules have excitation wavelengths that are within 15 nm of one another.   
     
     
         2 . The labeled oligonucleotide according to  claim 1 , wherein at least two of said photometric molecules produce a detectable change in signal when at least two of said at least two photometric molecules are separated from one another. 
     
     
         3 . The labeled oligonucleotide according to  claim 2 , wherein said at least two photometric molecules are separated from one another when said labeled oligonucleotide is cleaved at a position between said at least two photometric molecules. 
     
     
         4 . The labeled oligonucleotide according to  claim 2 , wherein said at least two photometric molecules are separated from one another when said labeled oligonucleotide hybridizes to the target sequence. 
     
     
         5 . The labeled oligonucleotide according to  claim 2 , wherein said at least two photometric molecules are separated from one another when said labeled oligonucleotide is incorporated into a polynucleotide. 
     
     
         6 . The labeled oligonucleotide according to  claim 2 , wherein said two photometric molecules is separated from one another when said two molecules are cleaved from said labeled oligonucleotide. 
     
     
         7 . The labeled oligonucleotide according to  claim 1 , wherein said at least two photometric labeling molecules are fluorescent molecules. 
     
     
         8 . The labeled oligonucleotide according to  claim 1 , wherein any pair of said photometric labeling molecules on said oligonucleotide is separated from one another by about 3 to about 60 nucleotides. 
     
     
         9 . The labeled oligonucleotide according to  claim 1 , wherein any pair of said photometric labeling molecules on said oligonucleotide is separated from one another by about 12 to about 35 nucleotides. 
     
     
         10 . The labeled oligonucleotide according to  claim 1 , wherein any pair of said photometric labeling molecules on said oligonucleotide is separated from one another by about 15 to about 25 nucleotides. 
     
     
         11 . The labeled oligonucleotide according to  claim 1 , further including:
 at least one quenching molecule attached to said oligonucleotide, wherein   said quenching molecule quenches a signal produced by said at least two photometric labeling molecules when said at least two photometric labeling molecules are attached to said oligonucleotide.   
     
     
         12 . The labeled according to  claim 1 , wherein at least one of said at least two photometric labeling molecules is a fluorescent dye, selected from the group consisting of a dye that has delocalized positive charge, a dye that has a delocalized negative charge, and a dye that has an equal number of positive and negative charges. 
     
     
         13 . The labeled oligonucleotide according to  claim 1 , wherein at least one of said at least two photometric labeling molecules is a dye selected from the group consisting of BODIPY, a cyanine dye with delocalized positive charge and a zwiterionic cyanine dye. 
     
     
         14 . The labeled oligonucleotide according to  claim 1 , wherein at least one of said at least two labeling molecules is xanthene dye molecule. 
     
     
         15 . The labeled oligonucleotide according to  claim 1 , wherein at least one of said at least two photometric labeling molecules is a non-sulfonated cyanine dye molecule. 
     
     
         16 . The labeled oligonucleotides according to  claim 1 , wherein at least one of said at least two photometric labeling molecules is a dye selected from the group consisting of CR110, CR6G, TAMRA and ROX. 
     
     
         17 . The labeled oligonucleotide according to  claim 1 , wherein said oligonucleotide further includes at least one minor groove binder (MGB). 
     
     
         18 . The labeled oligonucleotide according to  claim 1 , including:
 at least one Guanidine nucleotide in said oligonucleotide, wherein   at least one of said at least two photometric labeling molecule is positioned near enough to said Guanidine nucleotide such that a signal produced by said at least one labeling molecule is quenched when said oligonucleotide is not hybridized to said target oligonucleotide.   
     
     
         19 . The labeled oligonucleotide according to  claim 1 , wherein said oligonucleotide has a 5′-end and a 3′-end; wherein one of said at least two photometric labeling molecules is attached to the said 5′-end of said oligonucleotide; wherein another of said at least two photometric labeling molecules is attached to the 3′-end of said oligonucleotide. 
     
     
         20 . The labeled oligonucleotide according to  claim 16 , wherein said photometric labeling molecules attached to said 5′-end and said 3′-end of said oligonucleotide molecule are attached to said oligonucleotide by flexible aliphatic linkers. 
     
     
         21 . The oligonucleotide according to  claim 1 , wherein said oligonucleotide sequence is substantially devoid of internal secondary structure. 
     
     
         22 . The oligonucleotide according to  claim 1 , wherein said oligonucleotide is suitable for use as a probe. 
     
     
         23 . The oligonucleotide according to  claim 1 , wherein said oligonucleotide is suitable for use as a primer in a polynucleotide amplification reaction. 
     
     
         24 . The oligonucleotide according to  claim 23 , wherein said polynucleotide amplification reaction is selected from the group consisting of PCR, multiplex PCR; real time PCR, quantitative PCR and real time quantitative PCR. 
     
     
         25 . A method of labeling an oligonucleotide, comprising:
 providing an oligonucleotide sequence that hybridizes to a target polynucleotide sequence; and   attaching at least two photometric labeling molecules to said oligonucleotide, wherein said at least two photometric labeling molecules have excitation wavelengths that are within 15 nm of one another.   
     
     
         26 . The method of labeling an oligonucleotide according to  claim 25 , wherein said at least two photometric labeling molecules are fluorescent molecules. 
     
     
         27 . The method of labeling an oligonucleotide according to  claim 25 , wherein any pair of said photometric labeling molecules on said oligonucleotide is separated from one another by about 3 to about 60 nucleotides. 
     
     
         28 . The method of labeling an oligonucleotide according to  claim 25 , wherein any pair of said photometric labeling molecules on said oligonucleotide is separated from one another by about 12 to about 35 nucleotides. 
     
     
         29 . The method of labeling an oligonucleotide according to  claim 25 , wherein any pair of said photometric labeling molecules on said oligonucleotide is separated from one another by about 15 to about 25 nucleotides. 
     
     
         30 . The method of labeling an oligonucleotide according to  claim 25 , further including:
 attaching at least one quenching molecule to said oligonucleotide, wherein said quenching molecule quenches a signal produced by said at least two photometric labeling molecules when said at least two photometric labeling molecules are attached to said oligonucleotide.   
     
     
         31 . The method of labeling an oligonucleotide according to  claim 25 , wherein at least one of said at least two photometric labeling molecules is a fluorescent dye, selected from the group consisting of a dye that has delocalized positive charge, a dye that has a delocalized negative charge, and a dye that has an equal number of positive and negative charges. 
     
     
         32 . The method of labeling an oligonucleotide according to  claim 25 , wherein at least one of said at least two photometric labeling molecules is a dye selected from the group consisting of BODIPY, a cyanine dye with delocalized positive charge and a zwiterionic cyanine dye. 
     
     
         33 . The method of labeling an oligonucleotide according to  claim 25 , wherein at least one of said at least two labeling molecules is xanthene dye molecule. 
     
     
         34 . The method of labeling an oligonucleotide according to  claim 25 , wherein at least one of said at least two photometric labeling molecules is a non-sulfonated cyanine dye molecule. 
     
     
         35 . The method of labeling an oligonucleotide according to  claim 25 , wherein at least one of said at least two photometric labeling molecules is a dye selected from the group consisting of CR110, CR6G, TAMRA and ROX. 
     
     
         36 . The method of labeling an oligonucleotide according to  claim 25 , wherein said method further includes attaching at least one minor groove binder (MGB) to said oligonucleotide. 
     
     
         37 . The method of labeling an oligonucleotide according to  claim 25 , including providing at least one Guanidine nucleotide in said oligonucleotide, wherein at least one of said at least two photometric labeling molecule is positioned near enough to said Guanidine nucleotide such that a signal produced by said at least one labeling molecule is quenched when said oligonucleotide is not hybridized to said target oligonucleotide. 
     
     
         38 . The method of labeling an oligonucleotide according to  claim 25 , wherein said oligonucleotide has a 5′-end and a 3′-end further comprising:
 attaching one of said at least two photometric labeling molecules to the said 5′-end of said oligonucleotide; and attaching another of said at least two photometric labeling molecules to the 3′-end of said oligonucleotide. 
 
     
     
         39 . The method of labeling an oligonucleotide according to  claim 25 , wherein said photometric labeling molecules attached to said 5′-end and said 3′-end of said oligonucleotide molecule are attached to said oligonucleotide by flexible aliphatic linkers. 
     
     
         40 . The method of labeling an oligonucleotide according to  claim 25 , wherein said oligonucleotide sequence is substantially devoid of internal secondary structure. 
     
     
         41 . The method of labeling an oligonucleotide according to  claim 25 , further comprising using said oligonucleotide as a primer probe. 
     
     
         42 . The method of labeling an oligonucleotide according to  claim 25 , further comprising using said oligonucleotide as a primer in a polynucleotide amplification reaction. 
     
     
         43 . A kit for labeling an oligonucleotide, comprising:
 an oligonucleotide sequence suitable for hybridizing to a target polynucleotide sequence; and   at least two photometric labeling molecules suitable for attachment to said oligonucleotide; wherein said at least two photometric labeling molecules have excitation wavelengths that are within 15 nm of one another.   
     
     
         44 . The kit for labeling an oligonucleotide according to  claim 43 , wherein at least one of said at least two photometric labeling molecule are selected from the group consisting of a dye that has delocalized positive charge, a dye that has a delocalized negative charge, and a dye that has an equal number of positive and negative charges. 
     
     
         45 . The kit for labeling an oligonucleotide according to  claim 43 , wherein at least one of said at least two photometric labeling molecule are selected from the group consisting of BODIPY, a cyanine dye with delocalized positive charge and a zwiterionic cyanine dye. 
     
     
         46 . The kit for labeling an oligonucleotide according to  claim 43 , wherein at least one of said at least two photometric labeling molecules is a xanthene dye molecule. 
     
     
         47 . The kit for labeling an oligonucleotide according to  claim 43 , wherein at least one of said at least two photometric labeling molecule is a non-sulfonated cyanine dye molecule. 
     
     
         48 . The kit for labeling an oligonucleotide according to  claim 43 , wherein at least one of said at least two photometric labeling molecules is a dye selected from the group consisting of CR110, CR6G, TAMRA and ROX. 
     
     
         49 . The kit for labeling an oligonucleotide according to  claim 43 , wherein said kit further includes a minor groove binder (MOB). 
     
     
         50 . A method of using a labeled oligonucleotide comprising the steps of:
 providing a labeled oligonucleotide, wherein said labeled oligonucleotide includes an oligonucleotide sequence suitable for hybridizing to a target polynucleotide sequence; at least two photometric labeling molecules, wherein said at least two photometric labeling molecules have excitation wavelengths that are within 15 nm of one another and, wherein said photometric labeling molecules are attached to said oligonucleotide;   contacting said labeled oligonucleotide with a sample.   
     
     
         51 . The method of using a labeled oligonucleotide according to  claim 50 , wherein said sample is selected from the group consisting of tissue extract, cell extract, bodily fluid, in vitro nucleic acid synthesis reaction, and PCR reaction mixture. 
     
     
         52 . A method of using a labeled oligonucleotide according to  claim 50 , wherein said labeled oligonucleotide is used in a nucleic acid amplification reactions wherein the reaction is selected from the group consisting of PCR, Multiplex PCR, Real Time PCR, Quantitative PCR, and Real Time Quantitative PCR. 
     
     
         53 . A method of using a labeled oligonucleotide according to  claim 50 , wherein said oligonucleotide is a probe for identifying the presence of a target sequence of nucleic acid polymer in the sample. 
     
     
         54 . A method of using a labeled oligonucleotide according to  claim 50 , wherein said sample is applied to a nucleic acid chip.