Mobility-Modifying Cyanine Dyes

Details Mobility-Modifying Cyanine Dyes Mobility-Modifying Cyanine Dyes

2000-01-04

2004-04-06

McKane, Joseph K. (Department: 1626)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C430S581000

Reexamination Certificate

active

06716994

ABSTRACT:

1. FIELD OF THE INVENTION
The present invention relates generally to fluorescent dye compounds that are useful as molecular probes. In particular, the present invention relates to fluorescent cyanine dye compounds that are mobility modified for use in nucleic acid sequencing reactions.
2. BACKGROUND OF THE INVENTION
The advent of automated four-color Sanger-type DNA sequencing has revolutionized the speed with which stretches of DNA can be reliably sequenced. In four-color Sanger-type DNA sequencing, a single-stranded target DNA of interest is hybridized with a complementary primer and the primer enzymatically extended with a DNA polymerase in the presence of a mixture of 2′-deoxyribonucleotides capable of supporting continuous primer extension (e.g., dATP, dGTP, dCTP and dTTP or dUTP) and a mixture of four labeled terminators. Each of the terminators is labeled with a different, spectrally distinguishable fluorescent label and terminates primer extension at a single type of template nucleotide. A mixture of terminators is used such that a termination event is achieved at each type of template nucleotide. The product of this primer extension or sequencing reaction is a nested set of labeled primer extension products in which the 3′-terminal nucleotide is identifiable by the color of its fluorescent label. These products are then electrophoretically separated, typically in a single gel lane or capillary, and the sequence of the target DNA determined from the colors of the resultant electrophoresis bands.
To avoid ambiguities in determining the sequence of the target DNA, the dyes used to label the primer extension products should either impart no electrophoretic mobility shifts on the products or impart uniform mobility shifts. However, in most instances, different types of dyes impart vastly different electrophoretic mobility shifts. Since the dyes must be spectrally distinguishable from one another, dyes having different structures, and hence quite different imparted electrophoretic mobility shifts, must be used. While sets of terminators that impart primer extension products with similar mobility shifts are available, rationally designing such sets of “mobility matched” terminators is currently virtually impossible. Rather, the sets are obtained through empirical trial and error. To date, no methods exists whereby one can predictably alter the electrophoretic mobilities imparted by terminators labeled with desirable dyes without altering the spectral properties of the dyes and/or jeopardizing the abilities of the labeled terminators to act as substrates for polymerizing enzymes. Accordingly, these are objects of the present invention.
3. SUMMARY OF THE INVENTION
These and other objects are furnished by the present invention, which in one aspect provides cyanine dye compounds having a mobility-modifying moiety that permits the electrophoretic mobilities of polynucleotides labeled with the dyes to be adjusted or tuned in a predictable fashion.
Cyanine dyes are a well-recognized class of fluorescent molecules which generally comprise first and second parent heteroaromatic ring systems covalently linked together via a methine, polymethine or cyclic alkylene bridge. The dyes may be homodimers, in which the first and second parent heteroaromatic ring systems are both members of the same class, or they may be heterodimers, in which the first and second parent heteroaromatic ring systems are both members of different classes. The parent ring systems may be optionally substituted with one or more substituents which can serve to alter the spectral, chemical and/or physical properties of the dyes.
The present invention concerns the class of cyanine dyes in which both parent heteroaromatic rings belong to the class of rings generally referred to as benzazoles/benzazoliums. The mobility-modifying cyanine dyes of the invention generally comprise: (i) a first parent benzazole/benzazolium heteroaromatic ring system that is substituted at the heteroaromatic ring nitrogen with a linking moiety; (ii) a second parent benzazole/benzazolium heteroaromatic ring system that is substituted at the heteroaromatic ring nitrogen with a mobility-modifying moiety; and (iii) a bridge linking the first and second parent benzazole/benzazolium rings via their respective C-2 carbons. The first and second parent benzazole/benzazolium ring systems may be the same or different, and may be optionally substituted with one or more of the same or different substituent groups. Preferably, both parent benzazole/benzazolium ring systems are the same or different substituted or unsubstituted indoline/indolinium ring systems. Depending upon the particular application, the linking moiety can be used to conjugate, preferably by way of covalent attachment, the mobility-modifying dyes of the invention to other molecules or substances.
Quite significantly, since the mobility-modified and linking moieties are located at opposing ends of the cyanine dye (i.e., on different heteroaromatic rings), nucleosides/tides and/or nucleoside/tide analogs labeled with the mobility-modifying cyanine dyes of the invention, e.g., labeled 2′-deoxyribonucleoside-5′-triphosphates and labeled terminating ribonucleoside-5′-triphosphates (e.g., 2′,3′-dideoxyribonucleoside-5′-triphosphates), retain high activity as substrates for DNA polymerizing enzymes, making the mobility-modifying dyes ideal for use in fluorescence-based nucleic acid sequencing applications. Moreover, since the electrophoretic mobilities of polynucleotides labeled with the mobility-modifying dyes can be predictably tuned to match those labeled with other dyes, the mobility-modifying dyes of the invention are ideal for use in 4-color fluorescence-based nucleic acid sequencing reactions, as sets of dyes having matched mobilities in addition to desirable spectral and biological properties can be readily obtained.
Virtually any known cyanine dye can be mobility-modified according to the principles of the invention. Thus, parent heteroaromatic ring systems of which the dyes of the invention can be comprised include, but are not limited to, the substituted and unsubstituted benzazole/benzazolium rings comprising the cyanine, merocyanine and styryl dyes described in U.S. Pat. Nos. 5,486,616, 5,569,587, 5,569,766 and 5,627,027; the substituted and unsubstituted benzazole/benzazolium rings comprising the asymmetric cyanine dyes described in U.S. Pat. Nos. 5,321,130, 5,410,030, 5,436,134, 5,534,416, 5,582,977, 5,658,751, 5,656,449, and 5,863,753; and the substituted and unsubstituted benzazole/benzazolium rings comprising the various sulfonated cyanine dyes described in Tu et al., 1998,
Nucl. Acids Res.
26(11):2797-2802, the disclosures of which are incorporated herein by reference. Additional substituted and unsubstituted benzazole/benzazolium ring systems of which the mobility-modifying cyanine dyes may be comprised are described in Brooker et al., 1945, “Absorption Spectra of Dyes with Heteroaromatic Nuclei—Color and Constitution. Part VII. Intepretation of Absorptions of Dyes Containing Heterocyclic Nuclei of Different Basicities,”
J. Am. Chem. Soc.
67:1875-1889 (in particular at page 1878), the disclosure of which is incorporated herein by reference.
The mobility-modifying moiety comprises a pendant group bearing a plurality of charges through substitution with one or more of the same or different charged substituents. The pendant group can be any moiety capable of being substituted with the desired number of charged substituents, but is typically a group having the structure —D-D′, where D is (C
1
-C
6
) alkyldiyl or 2-6 membered heteroalkyldiyl; and D′ is (C
1
-C
6
) alkyl, 2-6 membered heteroalkyl, (C
5
-C
14
) aryl, (C
5
-C
14
) arylaryl, 5-14 membered heteroaryl or 5-14 membered heteroaryl-heteroaryl. When D is heteroalkyldiyl, it must be attached to the benzazole/benzazolium ring nitrogen atom via an alkyldiyl group. Preferred amongst the various D groups is (C
1
-C
6
) alkyleno, particularly (C
1
-C
6
) alkanos su

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