Fluorescent nucleobase conjugates having anionic linkers

Abstract

Provided are nucleotide-dye conjugates and related compounds in which a dye is linked to a nucleobase directly or indirectly by an anionic linker. The anionic character of the linker is provided by one or more anionic moieties which are present in the linker, such as phosphate, phosphonate, sulfonate, and carboxylate groups. When the dye is a provided as a donor/acceptor dye pair, the anionic linker can be located between the donor and the acceptor, or between the nucleobase and either the donor or acceptor, or both. In one embodiment, conjugates of the invention provide enhanced electrophoretic mobility characteristics to sequencing fragments, e.g., for dideoxy sequencing using labeled terminators.

1 . A conjugate comprising a dye labeled nucleobase of the form:
 (1) B-L-D, wherein B is a nucleobase, L is an anionic linker, and D comprises at least one fluorescent dye, or   (2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 are linkers such that at least one of L1 and L2 is an anionic linker, and D1 and D2 are members of a fluorescent donor/acceptor pair, such that one of D1 and D2 is a donor dye capable of absorbing light at a first wavelength and emitting energy in response thereto, and the other of D1 and D2 is an acceptor dye capable of absorbing energy emitted by the donor dye and fluorescing at a second wavelength in response thereto; and   wherein L or at least one of L1 and L2 comprises a sulfonated benzene moiety.   
     
     
         2 . The conjugate of  claim 1  wherein the dye-labeled nucleobase is of the form B-L-D. 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The conjugate of  claim 2  wherein L additionally comprises an anionic phosphate moiety. 
     
     
         6 . The conjugate of  claim 5  wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L within a chain of linker atoms that connect B to D. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The conjugate of  claim 2  wherein L comprises a carboxylic acid moiety. 
     
     
         10 . The conjugate of  claim 9  wherein the carboxylic acid moiety is a carboxyl benzene moiety. 
     
     
         11 . The conjugate of  claim 1  wherein L comprises 4 to 20 chain atoms. 
     
     
         12 . The conjugate of  claim 1  wherein D comprises at least one xanthene, rhodamine, dibenzorhodamine, fluorescein, [8,9]benzophenoxazine, cyanine, phthalocyanine, squaraine, or bodipy dye. 
     
     
         13 . The conjugate of  claim 12  wherein D comprises at least one fluorescein or rhodamine. 
     
     
         14 . The conjugate of  claim 1  wherein B comprises adenine, 7-deazaadenine, 7-deaza-8-azaadenine, cytosine, guanine, 7-deazaguanine, 7-deaza-8-azaguanine, thymine, uracil, or inosine. 
     
     
         15 . The conjugate of  claim 1  wherein the labeled nucleobase is of the form B-L1-D1-L2-D2. 
     
     
         16 . The conjugate of  claim 15  wherein L1 is an anionic linker. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . The conjugate of  claim 16  wherein L1 comprises an anionic phosphate moiety. 
     
     
         20 . The conjugate of  claim 19  wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L1 within a chain of linker atoms that connect B to D1. 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . The conjugate of  claim 16  wherein L1 comprises a carboxylic acid moiety. 
     
     
         24 . The conjugate of  claim 23  wherein the carboxylic acid moiety is a carboxy benzene moiety. 
     
     
         25 . The conjugate of  claim 1  wherein L1 comprises 4 to 20 linker chain atoms. 
     
     
         26 . The conjugate of  claim 1 , wherein at least one of D1 or D2 comprises a xanthene, rhodamine, dibenzorhodamine, fluorescein, [8,9]benzophenoxazine, cyanine, phthalocyanine, or squaraine dye. 
     
     
         27 . The conjugate of  claim 15  wherein L2 comprises the sulfonated benzene moiety. 
     
     
         28 . The conjugate of  claim 27  wherein D1-L2-D2 comprises structure (c) below:
 (c) -D1-R 28 R 22 R 28 -D2   wherein:   R 22  is phena-1,4-diyl, and   R 28  is —R 29 Z 2 —C(O)— wherein R 29  is C 1 -C 5  alkyldiyl, and Z 2  is NH, S, or O.   
     
     
         29 . The conjugate of  claim 1 , wherein D1 is a donor dye and D2 is an acceptor dye. 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . The conjugate of  claim 28  wherein R 29  is CH 2 , and Z 2  is NH. 
     
     
         33 . The conjugate of  claim 1  wherein L2 comprises up to 20 linker chain atoms. 
     
     
         34 .- 45 . (canceled) 
     
     
         46 . The conjugate of  claim 15  wherein L2 is an anionic linker and L1 is not an anionic linker. 
     
     
         47 .- 57 . (canceled) 
     
     
         58 . The conjugate of  claim 46  wherein L1 comprises one of the following moieties:
 —CCCH 2 NH—, —C≡CCH 2 NHC(O)(CH 2 ) 5 NH—, —C═CC(O)NH(CH 2 ) 5 NH—,   —C≡CCH 2 OCH 2 CH 2 NH—, —C≡CCH 2 OCH 2 CH 2 OCH 2 CH 2 NH—, —C≡C—CH 2 OCH 2 CH 2 —NH—,   and —C≡C(p-C 4 H 6 )OCH 2 CH 2 NH—.   
     
     
         59 . A labeled nucleoside triphosphate comprising a conjugate of  claim 1 . 
     
     
         60 . The labeled nucleoside triphosphate of  claim 59  which is not 3′-extendable. 
     
     
         61 . The labeled nucleoside triphosphate of  claim 60  which is a 2′,3′-dideoxynucleotide or 3′-fluoro-2′,3′-dideoxynucleotide. 
     
     
         62 . The labeled nucleoside triphosphate of  claim 59  which contains a 3′-hydroxyl group. 
     
     
         63 . A polynucleotide comprising a conjugate of  claim 1 . 
     
     
         64 . The polynucleotide of  claim 63  wherein the conjugate is located on a 3′ terminal nucleotide subunit. 
     
     
         65 . The polynucleotide of  claim 64  wherein the 3′ terminal nucleotide subunit is not extendable. 
     
     
         66 . The polynucleotide of  claim 65  wherein the 3′ terminal nucleotide subunit is a 2′,3′-dideoxynucleotide or 3′-fluoro-2′,3′-dideoxynucleotide. 
     
     
         67 . The polynucleotide of  claim 64  which contains a 3′-hydroxyl group. 
     
     
         68 . The polynucleotide of  claim 63  wherein the conjugate is located on a non-terminal nucleotide subunit. 
     
     
         69 . A mixture comprising a plurality of different-sequence polynucleotides, wherein at least one polynucleotide contains a conjugate of  claim 1 . 
     
     
         70 . The mixture of  claim 69  which comprises at least two different-sequence polynucleotides that each contain a conjugate of  claim 1 , such that each conjugate attached to the different-sequence polynucleotides may be the same or different. 
     
     
         71 . The mixture of  claim 69  which comprises four classes of polynucleotides, wherein the polynucleotides in each class terminate with a distinct terminator subunit type that contains a distinct conjugate of  claim 1  to identify the polynucleotides in that class. 
     
     
         72 . A nucleic acid sequencing kit comprising at least one labeled nucleoside triphosphate of  claim 59  and one or more of the following components:
 a 3′-extendable primer,   a polymerase enzyme,   one or more 3′ extendable nucleotides which are not labeled with conjugate, and/or   a buffering agent.   
     
     
         73 . The kit of  claim 72  wherein at least one labeled nucleoside triphosphate is nonextendable. 
     
     
         74 . The kit of  claim 72  comprising four different labeled nucleoside triphosphates configured to be complementary to A, C, T and G, wherein each contains a distinct conjugate of  claim 1 . 
     
     
         75 . The kit of  claim 74  wherein the four different labeled nucleoside triphosphates are nonextendable. 
     
     
         76 . The kit of  claim 74  wherein the four different labeled nucleoside triphosphates are extendable ribonucleoside triphosphates. 
     
     
         77 . The kit of  claim 72  wherein the conjugate has the form B-L1-D1-L2-D2. 
     
     
         78 . The kit of  claim 77  wherein the donor dye of each of the four different labeled nucleoside triphosphates is the same. 
     
     
         79 . The kit of  claim 78  wherein the donor dye is an orthocarboxyfluorescein. 
     
     
         80 . The kit of  claim 78  wherein the donor dye is a 4,7-dichloro-orthocarboxyfluorescein. 
     
     
         81 . A method of forming a labeled polynucleotide strand, the method comprising reacting together a duplex polynucleotide comprising (i) a 3′-extendable strand which is hybridized to a complementary template strand having a 5′ overhang, (ii) a template-dependent polymerase enzyme and (iii) a labeled nucleoside triphosphate containing a conjugate of  claim 1 , under conditions effective to form a labeled polynucleotide containing the conjugate. 
     
     
         82 . A method of sequencing a target polynucleotide sequence, the method comprising
 forming four classes of polynucleotides which are complementary to a target polynucleotide sequence, by template-dependent primer extension, wherein the polynucleotides in each class terminate with a different terminator subunit type that contains a distinct conjugate of  claim 1  to identify the polynucleotides in that class, and   separating the polynucleotides of the four classes on the basis of size to obtain a mobility pattern, and determining the sequence of the target polynucleotide sequence from the mobility pattern.   
     
     
         83 . The method of  claim 82  wherein the terminator subunits are nonextendable. 
     
     
         84 . The method of  claim 82  wherein the terminator subunits contain a 3′-hydroxyl group. 
     
     
         85 . A method of identifying one or more polynucleotide(s), the method comprising
 forming one or more labeled different-sequence polynucleotides, wherein each different-sequence polynucleotide contains a unique conjugate of  claim 1 , and   separating the one or more labeled different-sequence polynucleotides by electrophoresis so as to separate different-sequence polynucleotides on the basis of size, and   identifying each different-sequence polynucleotide on the basis of its electrophoretic mobility and fluorescence signal.   
     
     
         86 . The conjugate of  claim 1  wherein L1 is not acetylenic.