Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-05-16
2001-04-17
Wilson, James O. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S001110, C536S004100, C536S017200, C536S017300, C536S017400, C536S018500, C536S018600, C536S023100, C536S025300, C536S025320, C536S025340, C536S029200
Reexamination Certificate
active
06218108
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides fluorescent nucleosides carrying polycyclic aromatic hydrocarbons such as anthracene, phenanthrene, pyrene, stilbene, tetracene or pentacene. The subject nucleosides may be synthesized using a C-glycosidic bond formation method employing organocadmium or organozinc derivatives of the aromatic compounds and coupling with a 1-&agr;-chlororibose or deoxyribose synthon. The &agr;-anomers of the coupling reaction may be epimerized to the &bgr;-anomers by acid-catalyzed equilibration. The fluorescent nucleosides act as DNA or RNA base analogs and can be incorporated into nucleic acids. Resultant fluorescently tagged nucleic acids are useful as probes for target nucleic acids in tissues, solutions or immobilized on membranes.
BACKGROUND OF THE INVENTION
A large number of non-natural analogues of DNA nucleosides have been synthesized in recent years. Changing the structure of the base moiety attached to deoxyribose has been a useful strategy for probing structure and function in DNA. For example, a number of base analogues have been used to test the importance of specific hydrogen bonding interactions which may be important for function of the natural nucleic acid bases.
1-3
Using this strategy, the importance of hydrogen bonding in stabilizing DNA and RNA structure, in protein interactions and in the fidelity of enzymatic DNA and RNA synthesis has been examined.
Modified DNA bases have also been synthesized with the purpose of serving as reporter groups in physical and biochemical studies of structure and function. Examples of reporter groups which have been attached to DNA bases include biotin
4
and digoxigenin groups
5
, spin-label groups
6
, and DNA-cleaving moieties
7
. Among the most prominent classes of reporters used in DNA are fluorescent-tagged DNA bases which can serve as probes in biophysical and biochemical studies
8
. In contrast to placement of such reporter groups at the end of a DNA strand using nonnucleotide linkers, the attachment of a reporter to a DNA base allows for placement and probing within a stretch of DNA. Such a strategy has found considerable practical use in fluorescence-based automated DNA sequencing.
9
An alternative approach to the conjugation of a fluorophore to a natural DNA base is the more direct modification of a DNA base itself to render it fluorescent. A number of modified DNA bases with useful fluorescence properties have been reported recently; among the most widely used nucleosides of this type are 2-aminopurine
10
and ethenoadenosine
11
.
DNA may be fluorescently tagged either enzymatically or synthetically. Enzymatic incorporation is carried out by use of nucleoside triphosphates carrying a given fluorophore. Incorporation into DNA by chemical methods is especially common. Chemical methods of incorporating fluorescent reagents into DNA is done by either of two methods: direct incorporation of a label which has been converted to a phosphoramidite derivative or incorporation of an amine into the oligonucleotide, followed by later derivatization with a fluorophore isothiocynate or NHS ester derivative.
The postsynthesis derivatizaton of DNA is typically inefficient, and requires steps beyond those of standard DNA synthesis as well as laborious purification steps. Direct incorporation of a label into DNA is attractive because standard DNA synthesis and purification steps are used. However, currently available reagents are quite expensive due to their cost of synthesis.
Another drawback to currently used fluorescent-tagged nucleic acids is the quenching phenomenon which occurs when multiple fluorescent tags are placed near each other. Thus, a nucleic acid with multiple fluorescent tags is often no brighter or even less bright than a nucleic acid with a single fluorescent tag.
In addition, it is commonly observed that a fluorescent tag is quenched by the act of attaching it to DNA. For example, it has been reported (Netzel, 1989 J. Am. Chem. Soc. 111:6966) that pyrene tags attached to a linker at the end of a DNA strand were quenched greatly (50-fold) in the DNA. Moreover, on binding a complementary sequence, the emission was quenched another ten-fold. This effect necessarily leads to lower sensitivity of detection.
Another limitation of commercially available fluorescent labels include their rapid photobleaching characteristics. As in fluorescence microscopy or blot hybridization, the practical brightness of a label depends on the time of integration of the emission signal. Fluorescein, for example, photobleaches quite rapidly in a DNA oligonucleotide, because of its complex structure (allowing greater reactivity) and because it is exposed to solution where more reactions occur. In addition, a label such as fluorescein is typically attached to DNA by flexible tethers. Measuring protein-DNA binding by time-resolved fluorescence an isotropy is often problematic since the fluorophore tumbles rapidly on its flexible chain.
The present invention overcomes many of the shortcomings associated with labeling nucleic acids. The present invention allows a pyrene, anthracene, phenanthrene, stilbene, tetracene or pentacene-derivatized nucleoside to be inserted within a DNA or RNA strand at any position and remain rigidly stacked within the helix. Because the fluorescent part of the label is situated as if it were a DNA base, the fluorescent groups are stacked neatly in the helix, and if placed adjacent to each other, interact with each other strongly, allowing for intense excimer emission.
The fluorescent nucleoside analogs of the present invention do not photobleach rapidly, making the labeled sample much longer lived and allowing the opportunity for measurements and study over a longer period. Moreover, since the preferred embodiment of the invention provides for &agr;-linkages to the fluorescent moiety, quenching by adjacent &bgr;-linked DNA bases is minimized. In addition, the direct attachment of the fluorescent moiety to the sugar residue in the nucleotide chain eliminates the flexible linker typically present in fluorescently labeled nucleic acids. This feature simplifies measurements such as time-resolved fluorescence anisotropy.
SUMMARY OF THE INVENTION
The present invention provides fluorescent nucleoside analogs carrying a polycyclic aromatic hydrocarbon such as anthracene, phenanthrene, pyrene, stilbene, tetracene or pentacene. The polycyclic aromatic hydrocarbon is attached to the C1 (1′) position of a sugar moiety such as ribose or deoxyribose by a carbon-carbon bond. The sugar moiety may be a hexose such as glucose or a pentose such as arabinose. In one embodiment of the invention, the aromatic hydrocarbon attached to the C1 carbon of a sugar moiety is phenanthrene. In another embodiment of the invention, the aromatic hydrocarbon attached to the C1 carbon of a sugar moiety is pyrene. In still another embodiment of the invention, the aromatic hydrocarbon attached to the C1 carbon of a sugar moiety is anthracene. In another embodiment of the invention, the aromatic hydrocarbon attached to the C1 carbon of a sugar moiety is stilbene. In another embodiment of the invention, the aromatic hydrocarbon attached to the C1 carbon of a sugar moiety is tetracene. In another embodiment of the invention, the aromatic hydrocarbon attached to the C1 carbon of a sugar moiety is pentacene.
The present invention also provides phosphoramidite derivatives of anthracene, phenanthrene, pyrene, stilbene, tetracene, or pentacene-derivatized nucleosides. The phosphoramidite derivatives are useful in the chemical synthesis of nucleic acids containing phenanthrene, anthracene, pyrene, stilbene, tetracene, or pentacene-derivatized nucleosides.
The present invention also provides intermediates useful in the synthesis of the subject nucleosides and subject phosphoramidite derivatives of anthracene, phenanthrene, pyrene, stilbene, tetracene or pentacene-derivatized nucleosides. The intermediates comprise an adduct of Hoffer's chlorosugar and the corresponding polycyclic aromatic hydrocarbon.
Another aspec
Research Corporation Technologies Inc.
Scully Scott Murphy & Presser
Wilson James O.
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