Oligonucleotides and Analogs Labeled with Energy Transfer Dyes

Abstract

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers facilitate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

1 .- 23 . (canceled) 
     
     
         24 . An energy transfer dye, comprising:
 a xanthene donor dye configured to absorb excitation energy at a first wavelength and transferring energy in response;   an acceptor dye configured to absorb the energy transferred by the donor dye, and fluorescing at a second wavelength in response; and   a non-nucleosidic linker linking the donor dye to the acceptor dye, wherein the non-nucleosidic linker is a.) configured to efficiently transfer energy from the donor dye to the acceptor dye, b.) is attached to the 4′ position of the xanthene donor dye, and c.) comprises a functional group selected from the group consisting of an alkene, diene, alkyne, a five membered ring and a six membered ring having at least one unsaturated bond or fused ring structure.   
     
     
         25 . The energy transfer dye of  claim 24 , wherein the acceptor dye is a xanthene or cyanine dye. 
     
     
         26 . The energy transfer dye of  claim 25 , wherein when the acceptor dye is a xanthene dye, then the non-nucleosidic linker is attached to the xanthene acceptor dye at a 4′, 5 or 6 ring position. 
     
     
         27 . The energy transfer dye of  claim 25 , wherein the acceptor dye is selected from the group consisting of 4,7 dichlorofluorescein dyes, asymmetric benzoxanthene dyes, 4,7, dichlororhodamine dyes, carboxyrhodamine dyes, N,N, N′,N′-tetramethylcarboxyrhodamine dyes, carboxy-X-rhodamine dyes, and cyanine dyes. 
     
     
         28 . The energy transfer dye of  claim 27 , wherein the acceptor dye is a 4,7-dichlororhodamine dye. 
     
     
         29 . The energy transfer dye of  claim 27 , wherein the acceptor dye is carboxy R110, carboxy R6G, Cy5, or is a dye having one of the following formulae: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein L is the position to which the non-nucleosidic linker attaches on the acceptor dye. 
       
     
     
         30 . The energy transfer dye of  claim 24 , wherein the donor dye is selected from the group consisting of carboxyfluorescein dyes, 4,7 dichlorofluorescein dyes, asymmetric benzoxanthene dyes, 4,7, dichlororhodamine dyes, carboxyrhodamine dyes, and N,N,N′,N′-tetramethylcarboxyrhodamine dyes. 
     
     
         31 . The energy transfer dye of  claim 30 , wherein the donor dye is a carboxyfluorescein dye, a carboxyrhodamine dye or a N,N,N′,N′-tetramethylcarboxyrhodamine dye. 
     
     
         32 . The energy transfer dye of  claim 24 , wherein the non-nucleosidic linker comprises a five membered ring or a six membered ring having at least one unsaturated bond or fused ring structure. 
     
     
         33 . The energy transfer dye of  claim 32 , wherein the five membered ring and six membered ring having at least one unsaturated bond or fused ring structure is selected from the group consisting of cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine, oxazine, indene, benzofuran, thionaphthene, indole and naphthalene. 
     
     
         34 . The energy transfer dye of  claim 24 , having a structure of the following formula: 
       
         
           
           
               
               
           
         
         wherein R 1 , R 2 , R 3  and R 4  are each, independently of one another, selected from hydrogen, methyl and ethyl, or alternatively R 1  and R 5 , R 2  and R 6 , R 3  and R 8  and/or R 4  and R 9  are taken together with the atoms to which they are bonded to form a 5, 6 or 7-membered ring; 
         R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 6  and R 7  and/or R 9  and R 10  are taken together with the atoms to which they are bonded to form a benzo group; 
         X 1  and X 3  are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy; 
         L is the non-nucleosidic linker; 
         R 11 , R 12 , R 13 , R 15 , and R 16  are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 12  and R 13  and/or R 15  and R 16  are taken together with the atoms to which they are bonded to form a benzo group; and 
         X 11 , X 12 , X 13 , X 14  and X 15  are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy. 
       
     
     
         35 . A fluorescently labelled reagent, comprising:
 a reagent selected from the group consisting of a nucleoside, nucleotide monophosphate, nucleotide diphosphate, nucleotide triphosphate, oligonucleotide, and oligonucleotide analog, wherein the reagent is modified to be linked to an energy transfer fluorescent dye; and   an energy transfer fluorescent dye attached to the reagent, wherein the energy transfer fluorescent dye comprises:
 i. a xanthene donor dye configured to absorb excitation energy at a first wavelength and transferring energy in response, 
 ii. an acceptor dye configured to absorb the energy transferred by the donor dye, and fluorescing at a second wavelength in response, and 
 iii. a non-nucleosidic linker linking the donor dye to the acceptor dye, wherein the non-nucleosidic linker is a.) configured to efficiently transfer energy from the donor dye to the acceptor dye, b.) is attached to the 4′ position of the xanthene donor dye, and c.) comprises a functional group selected from the group consisting of an alkene, diene, alkyne, a five membered ring and six membered ring having at least one unsaturated bond or fused ring structure. 
   
     
     
         36 . The fluorescently labelled reagent of  claim 35 , wherein the acceptor dye is a xanthene or cyanine dye. 
     
     
         37 . The fluorescently labelled reagent of  claim 35 , wherein the attachment of the labelled reagent to the energy transfer dye is at a 5 or 6 ring position of the xanthene donor dye. 
     
     
         38 . The fluorescently labelled reagent of  claim 35 , wherein the energy transfer dye is attached to the reagent via a linkage comprising an acetylenic amido group or alkenic amido group. 
     
     
         39 . The fluorescently labelled reagent of  claim 38 , wherein the acetylenic amido or alkenic amido linkage is selected from —C≡C—CH 2 —NH—C(O)—, 3-amino-1-propyn-1-yl, —C≡C—CH 2 —NH—C(O)—(CH 2 ) 5 —C(O)—, —C═CH—C(O)—NH—(CH 2 ) 5 —NH—C(O)— and —C≡C—CH 2 —O—CH 2 —CH 2 —NR—, where R is hydrogen, a protecting group, or alkyl. 
     
     
         40 . The fluorescently labelled reagent of  claim 36 , wherein when the acceptor dye is a xanthene dye, then the non-nucleosidic linker is attached to the xanthene acceptor dye at a 4′, 5 or 6 ring position. 
     
     
         41 . The labelled reagent of  claim 36 , wherein the acceptor dye is selected from the group consisting of 4,7 dichlorofluorescein dyes, asymmetric benzoxanthene dyes, 4,7 dichlororhodamine dyes, carboxyrhodamine dyes, N,N,N′,N′-tetramethylcarboxyrhodamine dyes, carboxy-X-rhodamine dyes, and cyanine dyes. 
     
     
         42 . The fluorescently labelled reagent of  claim 41 , wherein the acceptor dye is a 4,7-dichlororhodamine dye. 
     
     
         43 . The fluorescently labelled reagent of  claim 35 , wherein the donor dye is selected from the group consisting of carboxyfluorescein dyes, 4,7 dichlorofluorescein dyes, asymmetric benzoxanthene dyes, 4,7, dichlororhodamine dyes, carboxyrhodamine dyes, and N,N,N′,N′-tetramethylcarboxyrhodamine dyes. 
     
     
         44 . The fluorescently labelled reagent of  claim 35 , wherein the non-nucleosidic linker comprises a five membered ring or a six membered ring having at least one unsaturated bond or fused ring structure. 
     
     
         45 . The fluorescently labelled reagent of  claim 35 , wherein the energy transfer dye has a structure of the following formula: 
       
         
           
           
               
               
           
         
         wherein R 1 , R 2 , R 3  and R 4  are each, independently of one another, selected from hydrogen, methyl and ethyl, or alternatively R 1  and R 5 , R 2  and R 6 , R 3  and R 8  and/or R 4  and R 9  are taken together with the atoms to which they are bonded to form a 5, 6 or 7-membered ring; 
         R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 6  and R 7  and/or R 9  and R 10  are taken together with the atoms to which they are bonded to form a benzo group; 
         X 1  and X 3  are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy; 
         L is the non-nucleosidic linker; 
         R 11 , R 12 , R 13 , R 15  and R 16  are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 12  and R 13  and/or R 15  and R 16  are taken together with the atoms to which they are bonded to form a benzo group; and 
         X 11 , X 12 , X 13 , X 14  and X 15  are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, and alkoxy, with the proviso that one of R 8 , X 13  or X 14  comprises a linkage that covalently links the energy transfer dye to the fluorescently labelled reagent. 
       
     
     
         46 . The fluorescently labelled reagent of  claim 35 , wherein the fluorescently labelled reagent is a nucleotide monophosphate, nucleotide diphosphate, or nucleotide triphosphate. 
     
     
         47 . The fluorescently labelled reagent of  claim 46 , wherein the fluorescently labelled nucleotide is a deoxynucleotide or a dideoxynucleotide.