Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-30
2002-09-17
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06451530
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods of detecting hybridization between nucleic acids or protein
ucleic acid interactions. In particular this invention pertains to nucleic acid probes containing a fluorescent nucleotide analogue whose fluorescence level increases when the probe hybridizes to a target nucleic acid or when the probe is bound by a protein.
BACKGROUND OF THE INVENTION
One of the most specific molecular recognition events takes place when a strand of nucleic acid anneals (hybridizes) to its complement. A single-stranded oligonucleotide probe can find a complementary strand in the presence of a large excess of other nucleic acids. This process has facilitated the exploration of gene structure and organization, the measurement of gene expression and function, and the detection and characterization of a wide variety of pathologies.
Hybridization based assays typically require detection and/or quantification of a hybrid duplex formed between a probe nucleic acid and its corresponding target nucleic acid. However, because the measurable changes in the physical properties of nucleic acids that occur upon hybridization are rather small, such assays most frequently utilize labels attached either to the nucleic acid probes or to the target nucleic acids to detect the hybrid duplexes. After hybridization, typically the hybridized nucleic acids are immobilized (e.g., attached to a membrane) and the unhybridized nucleic acids are washed away. The immobilized label is then detected and/or quantitated to provide a measure of the hybrid duplex.
The requirement that unhybridized probes be separated from hybridized probes precludes the use of hybridization for real-time monitoring of nucleic acid syntheses or protein-nucleic acid interactions or for location specific nucleic acids in living cells. In addition, the need to immobilize hybrids on a solid surface limits sensitivity, since probes bind non-specifically to surfaces. While several schemes have been put forward for detecting specific nucleic acids in homogeneous solutions (see, e.g., Heller et al. European Patent Application 82303699.1, Morrison et al. (1989)
Anal. Biochem.,
183: 231-244, Cardullo et al. (1988)
Proc. Natl. Acad. Sci. USA,
85: 8790-8794, Morrison et al. (1993)
Biochem.,
32: 3095-3104, and Sixou et al. (1994)
Nucl. Acids Res.,
22: 662-668), these methods are typically unsuitable for real-time measurements or use in living cells.
Newer approaches for the detection of nucleic acid hybridizations and protein-nucleic acid interactions typically rely on energy transfer between a fluorophore and a quencher molecule or a second fluorophore (e.g., a fluorescence resonance energy transfer system). Thus, for example, a lumazine derivative has been used in conjunction with a bathophenanthroline-ruthenium complex as an energy transfer system in which the lumazine derivative acted as an energy donor and the ruthenium complex acted as an energy receptor. The lumazine derivative and ruthenium complex were attached to different nucleic acids. Energy transfer occurred when the two compounds were brought into proximity resulting in fluorescence. The system provided a mechanism for studying the interaction of molecules bearing the two groups (see, e.g., Bannwarth et al.,
Helvetica Chimica Acta.
(1991) 74: 1991-1999, Bannwarth et al. (1991),
Helvetica Chimica Acta.
74: 2000-2007, and Bannwarth et al., European Patent Application No. 0439036A2).
Another approach utilizes nucleic acid probes bearing a fluorophore and a quencher molecule. The probes were self-complementary and adopted a hairpin conformation in solution. The hairpin juxtaposed the fluorophore and the quencher thereby reducing or eliminating fluorescence of the fluorophore. When the probes hybridized to a target nucleic acid, they linearized, separating the fluorophore from the quencher molecule and thereby providing a fluorescent signal (see Tyagi and Kramer et al. (1996)
Nature Biotechnology,
14: 303-308).
Both of these approaches required a fluorescent compound and a second fluorophore or a quencher. Most fluorescent compounds, however, generally suffer the disadvantage that the fluorescent complexes and their binding moieties are relatively large. The presence of large fluorescent labels and associated linkers may alter the mobility of the nucleic acid, either through a gel as in sequencing, or through various compartments of a cell.
In addition, the presence of these markers alters the interaction of the labeled nucleic acid with other molecules either through chemical interactions or through steric hindrance. The presence of these markers thus makes it difficult to study the interactions of DNA with other molecules such as other nucleic acids or proteins.
SUMMARY OF THE INVENTION
This invention provides new methods and compositions for the detection of nucleic acid interactions with other nucleic acids or with proteins. The methods and compositions utilize fluorescent nucleotide analogs as fluorescent moieties and thus do not suffer from the limitations described above.
The methods of this invention generally utilize a nucleic acid (e.g., an oligonucleotide) that contains one or more fluorescent nucleotide analogues. The fluorescence of the nucleotide analogues is quenched (reduced) when they are incorporated into the oligonucleotide (see, e.g., U.S. Pat. No. 5,525,711 and Hawkins et al. (1995)
Nucl. Acids Res.,
23: 2872-2880. However, when the fluorescent nucleotide analogue is removed from the quenching influence of neighboring bases (e.g, present in a loop) fluorescence activity is partially or completely restored. Without being bound by a particular theory, it is believed that alteration of the normal conformation (e.g., base stacking) of the oligonucleotide at the location of the fluorescent nucleotide analogue reduces and/or eliminates the quench thereby causing an increase in fluorescence.
Thus, in one embodiment, this invention provides methods of detecting the presence, absence, or quantity of a target nucleic acid. The methods involve contacting the target nucleic acid with a nucleic acid probe where the nucleic acid probe comprises a fluorescent nucleotide located in the probe such that, when the probe hybridizes to the target nucleic acid, the fluorescent nucleotide is present in a loop that does not participate in complementary base pairing with a nucleotide of the target nucleic acid; and detecting the fluorescence produced by the fluorescent nucleotide, when said probe forms a hybrid duplex with said target nucleic acid. In one preferred embodiment, the loop ranges in length from about 1 to about 100 nucleotides when the probe hybridizes to said target nucleic acid. In particularly preferred probes, the loop is an insertion in said nucleic acid probe which is otherwise complementary to said target nucleic acid or to a contiguous subsequence of said target nucleic acid. In some preferred embodiments, the insertion is three nucleotides in length and comprises two nucleotides each adjacent to the fluorescent nucleotide. In particularly preferred embodiments, at least one nucleotide adjacent to the fluorescent nucleotide is a purine (e.g., adenosine), and in still more preferred embodiments, the fluorescent nucleotide is bordered by at least two adjacent purines (e.g., adenosine) in both the 5′ and 3′ direction. In a most preferred embodiment, the insertion is a single base insertion; the fluorescent nucleotide.
In yet another embodiment, the insertion is self-complementary and forms a hairpin in which the fluorescent nucleotide is present in the loop of said hairpin and does not participate in complementary base pairing. The nucleotides comprising the loop can be selected such that they are not complementary to the corresponding nucleotides of the target nucleic acid when said probe is hybridized to said target nucleic acid and where said probe is complementary to at least two non-contiguous subsequences of said target nucleic acid.
In another embodiment, the fluorescent nucleotide is present in a te
Brusca John S.
The United States of America as represented by the Department of
Townsend and Townsend / and Crew LLP
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